Mutants of the fungus neosartorya fischeri sesquiterpene synthase and methods of construction and use thereof

Abstract

The application relates to the technical field of bioengineering, and discloses a Fischerella sp. NfSS sesquiterpene synthase mutant, a construction method and application thereof. The Fischerella sp. NfSS sesquiterpene synthase mutant is obtained by mutating T at the 88th position, S at the 235th position, Q at the 654th position, Q at the 654th position, Q at the 654th position or F at the 637th position of the amino acid sequence shown in SEQ ID NO. 1 into G, A, A, I, R or W. The Q654I, Q654R and F637W mutants in the application exhibit significantly enhanced Sesterfisherol production capacity; the T88G, S235A and Q654A mutants exhibit obvious product spectrum changes, and can produce geraniol, geranial and oxidized monoterpene by-products in addition to the main product Sesterfisherol; the NfSS is changed from low-efficiency natural catalysis to high-efficiency controllable biosynthesis, and a new technical path is provided for industrialized biological manufacturing of sesquiterpene natural products.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a Feshiosartos disesquiterpene synthase mutant, its construction method, and its application. Background Technology

[0002] Sesterpenes are a diverse class of natural products in the terpenoid family, exhibiting significant biological activity. Their chemical skeleton consists of 25 carbon atoms and is formed by the catalysis of sesterterpene cyclases (STCs). They hold broad application prospects in medicine, chemical engineering, and other fields. Sesterpenes have shown significant pharmacological effects in antitumor, anti-inflammatory, and antibacterial activities, and their structural diversity makes them a research hotspot in synthetic chemistry and natural product chemistry. However, due to the low abundance of sesterpenes in nature and their reliance on complex plant extraction or difficult-to-scale biosynthesis, their production cannot meet industrial demand.

[0003] Currently, while chemical synthesis methods can be used to synthesize certain simple disesquiterpenes, they typically require harsh conditions, consume large amounts of energy, and generate significant amounts of harmful waste, making them unsuitable for large-scale green synthesis. On the other hand, naturally derived disesquiterpenes are costly to extract, have poor stability, and are significantly affected by environmental factors (such as season, climate, and soil conditions), thus severely limiting their production. These issues make the large-scale production of disesquiterpenes a current challenge. Therefore, exploring new, efficient, and sustainable production pathways, particularly the efficient biosynthesis of disesquiterpenes through microbial fermentation, is an important research direction in the field of natural products.

[0004] NfSS (N. fischeri-derived sesquiterpene synthase), a key enzyme in the synthesis of sesquiterpenes, has high application potential. NfSS is a chimeric bifunctional enzyme containing PT and TC domains, capable of catalyzing the production of sesquiterpenes from GFPP. During its catalytic process, the PT domain is responsible for substrate transfer, while the TC domain generates polycyclic structures through complex cyclization reactions. However, despite its crucial role in sesquiterpene synthesis, NfSS currently suffers from low catalytic efficiency, limited product diversity, and poor stability, which are the main technical challenges for its industrial application. Summary of the Invention

[0005] The purpose of this invention is to provide mutants of *Neosatoria fischeri* sesquiterpene synthase, their construction methods, and applications. The Q654I, Q654R, and F637W mutants exhibit significantly enhanced Sesterfisherol production capacity, especially Q654I and Q654R, which are approximately 63% and 59% higher than the wild type, respectively. The T88G, S235A, and Q654A mutants show obvious changes in their product profiles, producing not only the main product Sesterfisherol but also geraniol, geranilic acid, and oxidized monoterpenoid byproducts. This invention realizes the transformation of NfSS from inefficient natural catalysis to efficient and controllable biosynthesis, providing a new theoretical basis and technical pathway for the industrial biomanufacturing of sesquiterpene natural products.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a *Neosatoria freundii* sesquiterpene synthase mutant, wherein the *Neosatoria freundii* sesquiterpene synthase mutant is obtained by mutating the threonine T at position 88 of the amino acid sequence shown in SEQ ID NO.1 to glycine G, the serine S at position 235 to alanine A, the glutamine Q at position 654 to alanine A, the glutamine Q at position 654 to isoleucine I, the glutamine Q at position 654 to arginine R, or the phenylalanine F at position 637 to tryptophan W.

[0007] Furthermore, the Fessiformia disesquiterpene synthase mutant, using geraniol farnesyl pyrophosphate (GFPP) as a substrate, produced a product profile including sesquiterpene fish oil alcohol (Sesterfisherol), monoterpene geraniol (Geraniol), monoterpene geranic acid (Geranilic acid), and pulegone oxide (Pulegone oxide).

[0008] Secondly, the present invention provides the encoding gene for a sesquiterpene synthase mutant of *Fishia neosatula*.

[0009] Thirdly, the present invention provides a recombinant plasmid containing the encoding gene of the above-mentioned Feshiosartos disesquiterpene synthase mutant.

[0010] Furthermore, the method for constructing the recombinant plasmid is as follows: site-directed mutagenesis is performed on the amino acid sequence of the disesquiterpene synthase of *Neotrichum ferri*, and the coding gene is synthesized. BamHI and HindIII restriction sites are introduced at both ends of the coding gene, respectively, and the gene is cloned into the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-NfSS.

[0011] Fourthly, the present invention provides a genetically engineered bacterium expressing the above-mentioned Feshinosatoria disesquiterpene synthase mutant.

[0012] Furthermore, the genetically engineered bacterium is Escherichia coli.

[0013] Fifthly, the present invention provides the application of the above-mentioned Feshinosatoria disesquiterpene synthase mutant in the preparation of disesquiterpene fish oil Sesterfisherol.

[0014] Furthermore, compared with the wild type, the yield of sesesterfisherol from the Feshiosartobacter disesesterfisherol synthase mutant was increased by 30-63%.

[0015] The advantages and positive effects of the *Neosatoria ferrocephala* disesquiterpene synthase mutant, its construction method, and its application described in this invention are as follows: This invention conducted site-directed mutagenesis studies around key sites. The results showed that the Q654I, Q654R, and F637W mutants exhibited significantly enhanced Sesterfisherol production capacity, especially Q654I and Q654R, which increased by approximately 63% and 59% respectively compared to the wild type. The T88G, S235A, and Q654A mutants showed obvious changes in product profiles, producing not only the main product Sesterfisherol but also geraniol, geranilic acid, and oxidized monoterpenoid byproducts. This invention realizes the transformation of NfSS from inefficient natural catalysis to efficient and controllable biosynthesis, providing a new theoretical basis and technical path for the industrial biomanufacturing of disesquiterpenoid natural products.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is the first GC-MS product analysis of the NfSS mutant T88G in this embodiment of the invention, wherein (a) is the total ion chromatogram of the mutant; (b) is a magnified view of the 21-23 min interval; Figure 2 This is the second GC-MS product analysis of the NfSS mutant T88G in this embodiment of the invention, wherein (a) is Figure 1 (a) is a magnified view of the 10–11.5 min range; (b) is the mass spectrum of the corresponding monoterpene peak. Figure 3 This is the first GC-MS product analysis of the NfSS mutant S235A in this embodiment of the invention, wherein (a) is the total ion chromatogram of the mutant; and (b) is a magnified view of the 21-23 min interval. Figure 4 GC-MS product analysis of NfSS mutant S235A in this embodiment of the invention, wherein (a) is Figure 3 (a) is a magnified view of the 10-11.5 min range; (b) is the mass spectrum of the corresponding monoterpene peak. Figure 5This is the first GC-MS product analysis of the NfSS mutant Q654A in this embodiment of the invention, wherein (a) is the full scan chromatogram of the mutant; (b) is the magnified chromatogram of the 21-23 min interval; Figure 6 This is the second GC-MS product analysis of the NfSS mutant Q654A in this embodiment of the invention, wherein (a) is Figure 5 (a) is a magnified view of the 10.5-11.5 min range; (b) is the mass spectrum of the corresponding monoterpene peak. Figure 7 This invention presents a comparison of the Sesterfisherol yield of NfSS PT domain mutants with that of wild-type NfSS. (a) shows the Sesterfisherol yield of different NfSS PT domain mutants as peak area, compared with wild-type (WT) NfSS; (b) shows the corresponding heatmap, where A represents WT NfSS and B to M represent L512I to Q654R / F637W. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0021] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0022] In this application, NfSS refers to *Neosatoria fressiformis* sesquiterpene synthase. Neosartorya fischeri The abbreviation for sesterterpene synthase.

[0023] In this application NfSSThe gene originates from a fungal secondary metabolic gene cluster, with its NCBI gene ID NFIA_055500 (Gene ID: 4584613), GenBank accession number NW_001509760, protein sequence number XP_001258098.1 (SEQ ID NO.1), and FungiDB database accession number NFIA_055500. The gene is 2477 bp in length, encoding a protein of 764 amino acid residues with a theoretical molecular weight (MW) of approximately 87.60 kDa. The protein's accession number in the UniProtKB / Swiss-Prot database is A1DN30. Furthermore, its resolved three-dimensional crystal structure corresponds to the PDB number 8YLA, and the AlphaFold database predicted structure number is AF-A1DN30-F1.

[0024] SEQ ID NO.1: .

[0025] Example 1: Computer-aided screening and confirmation of key NfSS mutation sites By combining multiple sequence alignment, phylogenetic analysis, homology modeling, molecular docking, DUET stability prediction, and Missense3D structural analysis, potential functional residues were comprehensively screened.

[0026] The NfSS TC domain (1-331 aa) is the core region catalyzing the carbocation cascade cyclization of geranyl farnesyl pyrophosphate (GFPP) to form a sesquiterpene skeleton. Its active pocket is constructed by several key residues. T88G, located within the TC active center, has a side chain that regulates substrate folding conformation and the spatial localization of the carbocation intermediate, significantly influencing cyclization pathway selection. Mutations at this site can alter the substrate folding pattern within the active pocket, leading to changes in product type. S235A participates in the construction of the active pocket's polar environment; its hydroxyl side chain can form a local hydrogen bond network, affecting intermediate orientation and termination, and is therefore considered a crucial site for regulating product profiles. Overall, key residues in the TC domain synergistically regulate carbocation intermediate stability, local spatial confinement, and cyclization pathways, collectively determining the efficiency and product diversity of NfSS-catalyzed sesquiterpene skeleton formation.

[0027] In contrast, the PT domain (332-759 aa) of NfSS is primarily responsible for the chain elongation reaction of isoprene precursors such as IPP and DMAPP, and for forming linear intermediates such as GFPP. Key residues in this domain mainly participate in substrate binding, pyrophosphate stabilization, and Mg... 2+ The catalytic activity of the PT domain involves coordination and precursor chain conformation regulation, rather than direct participation in cyclization reactions within the TC domain. Molecular docking results show that F637W and Q654I / Q654R are located near the PT active site. F637W, an aromatic hydrophobic residue, may stabilize the substrate conformation and related intermediates through CH-π interactions and hydrophobic stacking. Q654I / Q654R, on the other hand, can regulate substrate localization and chain elongation efficiency through hydrogen bond networks and spatial constraints, significantly influencing the catalytic performance of the PT domain.

[0028] Based on the above key site analysis, and further combined with DUET saturation mutation stability prediction, Missense3D structural change analysis, and molecular docking binding energy screening, a systematic evaluation of potential advantageous mutants was conducted. The results showed (Table 1) that the Q654I / Q654R mutation resulted in a slight expansion of the active pocket, which may be beneficial for substrate binding and intermediate migration; while F637W showed a slight contraction of the pocket, suggesting that it may enhance local substrate binding ability.

[0029] Table 1. NfSS amino acid residue mutations screened by computer.

[0030] Further molecular docking analysis revealed that the binding energies of the Q654I / Q654R mutants to IPP or DMAPP were lower than those to the wild-type NfSS, suggesting that they may possess higher substrate affinity. Among them, Q654I, Q654R, and F637W exhibited superior binding energy parameters, suggesting that these mutations may improve precursor chain elongation efficiency and catalytic stability by optimizing the hydrogen bond network, hydrophobic interactions, and substrate spatial localization.

[0031] Based on the combined results of DUET stability prediction, Missense3D structural analysis, and molecular docking, Q654 and F637 are key residues that regulate the catalytic performance of NfSS. Among them, Q654I, Q654R, and F637W have high potential application value, as they can maintain overall structural stability and may improve substrate binding ability and catalytic efficiency; while T88 and S235 are more likely to participate in product spectrum regulation and cyclization pathway selection.

[0032] Example 2: Product Diversity Modification of NfSS I. Experimental Methods: 1. Key mutation sites for NfSS were identified: T88G, S235A, and Q654A.

[0033] 2. Plasmid extraction and purification: contain NfSS The specific steps for plasmid extraction and purification of mutant genes are as follows: ① Take PN Escherichia coli and inoculate it into 20 mL LB liquid medium at an inoculation ratio of 1%. Then add 0.1% kanamycin and place it in a shaker at 37 ℃ and incubate at 180 rpm for about 12 h.

[0034] ② Take 1.5 mL of the cultured bacterial solution and transfer it to a 2 mL centrifuge tube. Centrifuge at 19,942 × 10⁻⁶. g Centrifuge at 2000 rpm for 2 minutes, discard the supernatant, and collect the bacterial precipitate at the bottom.

[0035] ③ Add 250 μL of Solution Ⅰ / RNase suspension to the centrifuge tube and repeatedly pipette until the cells are completely resuspended and mixed.

[0036] ④ Add 250 μL Solution II to lyse the bacterial cells and release the DNA and proteins inside the cells. Gently invert the centrifuge tube 8–10 times until the solution becomes clear and transparent.

[0037] ⑤ Quickly add 350 μL Solution Ⅲ to denature and precipitate the impurity proteins, thereby removing them. Immediately invert the centrifuge tube until a white precipitate appears inside.

[0038] ⑥ At room temperature, at 16,990 × g Centrifuge at a speed of 10 min.

[0039] ⑦ Use a pipette to transfer the supernatant obtained after centrifugation to the adsorption column, and spray at 11,800 × 10⁻⁶ rpm. g Centrifuge at a speed of 1000 rpm for 1 minute, then discard the waste liquid in the collection tube.

[0040] ⑧ Add 500 μL of HEPES buffered calcium (HBC) solution to the adsorption column to neutralize the pH of the solution, at a concentration of 11,800 × 10⁻⁶. g Centrifuge for 1 min and discard the filtrate in the collection tube.

[0041] 9. Add 700 μL of DNA Wash buffer to wash away excess salt ions from the adsorption column at 11,800 × 10⁻⁶. g After centrifugation for 1 min, discard the filtrate. Repeat this washing step twice.

[0042] ⑩ With 19,940 × g Centrifuge at 19,940 × 10⁻⁶ for 2 min to completely remove residual water from the adsorption column; add 75 μL of Elution buffer or sterile water to the adsorption column, replace with a new collection tube, and let stand at room temperature for 2 min; finally, centrifuge at 19,940 × 10⁻⁶ for 2 min to completely remove residual water from the adsorption column; finally, centrifuge at 19,94 g Centrifuge at 2000 rpm for 2 min, collect the elution buffer containing plasmids, and after confirming that the DNA concentration is within acceptable limits, store in a -20 ℃ refrigerator for later use.

[0043] 3. PCR primer design and amplification: according to NfSS Single-point mutation primers were designed for the mutation sites (Table 2).

[0044] Table 2 Primers for T88G, S235A, and Q654A mutations

[0045] The PCR reaction system is shown in Table 3.

[0046] Table 3 PCR reaction system

[0047] The PCR reaction procedure is shown in Table 4.

[0048] Table 4 PCR reaction procedure

[0049] Since the template plasmid was present in the PCR reaction product, it was digested overnight at 37 °C using DpnI enzyme. The reaction system is shown in Table 5.

[0050] Table 5 Composition of DpnⅠ digestion reaction system

[0051] The reaction system was incubated at 37 °C for 1 h to ensure complete digestion of the template plasmid, followed by short-term heat inactivation at 65–80 °C to terminate enzyme activity. The product treated with DpnⅠ can be directly used in subsequent transformation steps, thereby significantly improving mutant screening efficiency and positive clone yield.

[0052] 4. Plasmid transformation and positive clone screening and identification: After enzyme digestion, the DpnI enzyme was inactivated in a 70 °C water bath. The plasmid was then transformed into *E. coli* DH5α cells, and the main process is as follows: ① Remove Escherichia coli DH5α cells from a -80 ℃ refrigerator and place them on ice for 10 min to allow them to completely thaw.

[0053] ② Transfer 10 μL of plasmid to E. coli DH5α cells, gently pipette and incubate on ice for 30 min.

[0054] ③ Heat shock in a water bath at 42 ℃ for 90 seconds, then place back in ice and let stand for 3 minutes.

[0055] ④ Add 800 μL of LB liquid culture medium. This step must be carried out in a clean bench. Then place it in a shaker and incubate at 37°C and 180 rpm for 2 h.

[0056] ⑤ After obtaining the bacterial culture, concentrate it appropriately, spread it evenly on LB solid plates containing 1 / 1000 kanamycin, and incubate it upside down in a 37 ℃ constant temperature incubator for 12-16 h.

[0057] ⑥ For each mutant, about 3 single colonies were selected and inoculated separately into 20 mL of LB liquid medium containing kanamycin resistance, and cultured at 37 ℃ with shaking (180 rpm) for 8-12 h.

[0058] ⑦ Extract plasmids from E. coli DH5α cells and perform agarose gel electrophoresis analysis. Select plasmids with bright target bands for sequencing. Then, based on the sequencing results, confirm the positive mutants for the next step of the experiment.

[0059] ⑧ The successfully mutated NfSS mutant plasmid and Pm plasmid were co-transformed into Escherichia coli DE3 cells for expression. The plasmid transformation steps were the same as those described in ①-⑤ above. The difference was that in ②, 5 μL of each of the two plasmids was gently mixed and 1 / 1000 spectinomycin was added to the LB solid plate medium in ⑤. Then, single colonies were selected and screened in liquid medium containing antibiotics to obtain bacterial culture. The bacterial culture was then mixed with 40% sterilized glycerol at a ratio of 1:1 in a clean bench and stored in a -80 ℃ freezer.

[0060] 5. GC-MS determination of catalytic activity and Sesterfisherol yield of NfSS mutant: GC-MS was used to detect and analyze sesesterfisherol and other products in the samples. A sample of fermentation products or enzyme reaction system was taken and an equal volume of n-hexane was added. n Liquid-liquid extraction was performed using Hexane, followed by thorough shaking and centrifugation to separate the layers (16,990 × 10⁻⁶). g Collect the organic phase (after 10 min) for analysis. If necessary, add an appropriate amount of anhydrous sodium sulfate for drying.

[0061] GC-MS analysis was performed using an Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer detector. The chromatographic column was an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The injection port temperature was set to 250 °C, using splitless injection mode. The temperature program was as follows: initial temperature 60 °C, held for 2 min, increased to 200 °C at 10 °C / min, then increased to 280 °C at 5 °C / min and held for 5 min. The carrier gas was high-purity helium (99.999%), with a flow rate of 1.0 mL / min.

[0062] Mass spectrometry was performed using an electron impact ionization (EI) source with an ionization energy of 70 eV, an ion source temperature of 230 °C, and a quadrupole temperature of 150 °C. The scanning range was [missing information]. m / z 40–500. The presence of Sesterfisherol was confirmed by comparing the retention time and mass spectrometry fragmentation characteristics of the target peak, and by comparing with the standard spectral library of the National Institute of Standards and Technology (NIST). Peak area was used as a relative quantitative indicator for comparing the yields of different samples.

[0063] The catalytic products were analyzed using GC-MS and qualitatively identified using total ion current (TIC) chromatograms. TIC chromatograms show the relationship between the total signal intensity of all ions in the sample and their retention time, providing a visual spectral representation for identifying different compounds. In this invention, by analyzing the TIC of the reaction products, the formation of the target product Sesterfisherol can be determined, and the amount and mass of the product produced under different reaction conditions can be compared. This analysis provides necessary data support for further research on the efficiency of enzyme-catalyzed reactions and optimization of catalytic conditions.

[0064] This invention employs a chromatographic peak area normalization method for semi-quantitative analysis of products. Under the same chromatographic detection conditions, the peak area corresponding to the target product brassinotriene is used as the evaluation index. By comparing the peak area of ​​the target product under different reaction systems and conditions, the differences in product formation and variation patterns are relatively assessed. This method is based on the positive correlation between chromatographic peak area and product concentration, and can effectively reflect the relative trend of product content changes.

[0065] II. Results Analysis: 1. Construction and validation of plasmids and engineered bacteria for NfSS mutants T88G, S235A, and Q654A: In this embodiment, three mutant plasmids of NfSS, namely T88G, S235A and Q654A, were constructed, and these mutants were transformed into Escherichia coli to construct recombinant engineered bacteria.

[0066] First, regarding NfSS Key amino acid residues of the gene were designed and site-directed mutagenesis was implemented. Specifically, the NfSS mutant T88G replaced Thr at the T88 site with Gly, the NfSS mutant S235A replaced Ser at the S235 site with Ala, and the NfSS mutant Q654A replaced Glu at the Q654 site with Ala. These mutants were all constructed using PCR amplification and seamless cloning technology.

[0067] To verify the correctness of these mutant constructions, the recombinant plasmids were sequenced using Sanger sequencing technology. Sequencing results showed that the sequences at the mutation sites in all mutant plasmids were consistent with expectations, and no unexpected mutations or frameshifts were found. Sequencing results for the NfSS mutant T88G showed that Thr at the T88 site was successfully replaced with Gly; sequencing results for the NfSS mutant S235A showed that Ser at the S235 site was successfully replaced with Ala; and sequencing results for the NfSS mutant Q654A showed that Glu at the Q654 site was successfully replaced with Ala. These sequencing results ensured the accuracy of the mutation sites.

[0068] After gene cloning, PCR verification, and sequencing confirmation, all mutant plasmids were transformed into *E. coli* BL21(DE3) competent cells, successfully constructing recombinant engineered bacteria. Subsequently, recombinant proteins of the NfSS mutant were obtained by inducing protein expression, and overexpression of the target protein was confirmed by SDS-PAGE. Finally, the mutant engineered bacteria were used for subsequent enzyme activity analysis and product detection.

[0069] 2. Product profile analysis results of the NfSS mutant: like Figure 1-6 The GC-MS results show that in the successfully constructed NfSS mutants, T88G, S235A, and Q654A not only catalyze the formation of the target product, sesquiterpene sesterfisherol, but also additionally produce monoterpenoid compounds, demonstrating a significant product spectrum expansion. Compared to the wild-type enzyme, these mutants, by increasing the active pocket volume and reducing steric hindrance, allow the substrate to exhibit a more flexible binding conformation within the active site, thereby altering the carbocation cascade cyclization pathway and forming new product types.

[0070] 3. NfSS mutant T88G: like Figure 1 As shown, the catalytic products of the NfSS mutant were analyzed by GC-MS to further verify its product diversity and catalytic properties. Figure 1 In the middle (a), the total ion current (TIC) chromatogram of the mutant fermentation product is shown. Multiple chromatographic peaks appear in the range of 10-23 min, indicating that the mutant has a rich metabolite spectrum. Figure 1 (b) is a magnified view of a local area. A distinct characteristic peak was detected at approximately 22.0 min. After comparison with the standard, the peak was confirmed to be the target product Sesterfisherol, indicating that the mutant still retains the ability to generate the target product.

[0071] Figure 2 Image (a) shows two new characteristic peaks in the range of approximately 11.0–11.5 min, which were identified as the monoterpene geraniol (C). 10 H 18 O, MW: 154.25 g / mol) and monoterpene geranic acid (C 10 H 16 O2, MW: 168.23 g / mol). Geraniol is a typical oxygen-containing monoterpene alcohol, while geranilic acid is its oxidation product. The generation of both indicates that the mutant expands the substrate binding pocket and changes the catalytic reaction pathway, thereby broadening the enzyme's product spectrum. Figure 2(b) shows the mass spectrum of the monoterpene product. Its molecular ion peaks appear near m / z 154 and 168, respectively, and it exhibits typical isoprene fragment ions such as m / z 69, 55 and 41, which preliminarily confirms its monoterpene skeleton structure.

[0072] NfSS is a chimeric bifunctional disesquiterpene synthase containing two domains, PT and TC, responsible for precursor chain elongation and cyclization, respectively. T88 is located in the TC domain and typically participates in C… 25 The circularization of the substrate GFPP and the stability of intermediates are enhanced. The NfSST88G mutation not only generates the main product Sesterfisherol, but also unexpectedly produces C... 10 Monoterpenoids—geraniol and geranic acid—were involved, suggesting that the mutation indirectly affects the catalytic product output of the NfSS PT domain. Possible mechanisms include: ① Imbalance between substrate localization and translocation regulation: The T88G mutation in the NfSS TC domain alters the spatial structure and flexibility of the active pocket, affecting the localization of the substrate GFPP within the TC domain. This may lead to the premature release of GFPP or part of the isoprene chain back into the PT domain before cyclization of the TC domain, thereby increasing the catalytic opportunity of the PT domain for short-chain products and generating C. 10 Monoterpenes.

[0073] ② Decreased stability of carbocation intermediates: The shrinkage of the side chain of T88G may reduce the stabilizing effect of the cyclization process on carbocation intermediates, leading to C 25 The chain cannot successfully complete the cascaded ring formation, and some substrates remain in the PT domain for a longer time or are prematurely dissociated to form monoterpenes.

[0074] ③ Enzyme conformation coupling effect: The PT and TC domains of bifunctional enzymes maintain the overall conformation and substrate orientation through interaction. T88G may have broken this interdomain coupling, increasing the probability of the PT domain generating short-chain products, while decreasing the catalytic efficiency of the TC domain, leading to the formation of monoterpene byproducts.

[0075] ④ Changes in cofactors and the microenvironment of the active pocket: The T88G mutation may alter the polarity distribution or hydrogen bond network of the TC domain, thereby indirectly affecting the Mg in the PT domain. 2+ Coordination or pyrophosphate group stability leads to C 10 The substrate is released midway and forms Geraniol and Geranic acid.

[0076] Therefore, the T88G mutation, by altering the local spatiality and flexibility of the TC domain, perturbs the synergistic effect between the PT and TC domains, increasing the probability of the PT domain generating short-chain monoterpenoid products. This indicates that the function of each domain in a bifunctional enzyme is highly dependent on its spatial conformation and interdomain interactions; mutation of a single domain can affect the entire catalytic process and product profile. This observation provides a theoretical basis for the engineering of NfSS to generate diverse products.

[0077] 4. NfSS mutant S235A: like Figure 3 As shown, GC-MS analysis was performed on the catalytic products of the NfSS mutant to evaluate the changes in its product spectrum and catalytic performance. Figure 3 In the middle (a), the total ion current (TIC) chromatogram of the mutant fermentation product is shown, which shows that there are multiple chromatographic peaks in the range of 10-23 min, indicating that the mutant has a relatively rich metabolite. Figure 3 (b) is a magnified view of a local area. A distinct characteristic peak was detected at approximately 22.0 min. After comparison with the standard, it was confirmed that this peak was the target sesquiterpene product Sesterfisherol, indicating that the mutant still retains its ability to catalyze the formation of the target product.

[0078] Figure 4 Image (a) shows a new characteristic peak at approximately 11.1 min, with significant abundance, indicating that the mutant, in addition to generating Sesterfisherol, can also catalyze the formation of additional monoterpene byproducts. According to mass spectrometry analysis, the molecular formula of this compound is C2. 10 H 16 O2 belongs to oxygen-containing monoterpenes. Figure 4 (b) shows the mass spectrum of this monoterpene, with its molecular ion peak appearing at m / z 168.0, consistent with the theoretical molecular weight of 168.1150. Characteristic fragment ions include m / z 83.0 (base peak), 69.0, 55.0, and 41.0, among which m / z 69.0 and 41.0 are typical fragments of isoprene unit fragmentation, further confirming its terpene skeleton structure; in addition, the ion peaks at m / z 101.0, 123.1, 138.8, and 153.0 correspond to oxygen-containing fragments formed by different fragmentation pathways.

[0079] Based on the analysis of retention time, molecular ion peaks, and characteristic fragment information, the byproduct is presumed to be an oxygen-containing monoterpene, possibly pulegone oxide or its structural analogue. This study demonstrates that the NfSS mutant significantly expands the product spectrum while maintaining the ability to synthesize the target product, proving that structural optimization to expand the active pocket can effectively regulate the enzyme's catalytic properties, and providing preliminary evidence for further enzyme engineering and mechanistic research.

[0080] The NfSS mutant S235A can produce the monoterpene compound pulegone oxide or its structural analogues, providing new insights into the catalytic mechanism of the TC domain. S235, located within the TC active pocket, originally participates in the spatial confinement and cyclization pathway regulation of the carbocation intermediate through side-chain hydrogen bonding. The S235A mutation replaces this Ser residue with the smaller Ala, resulting in an increase in the local space of the active pocket and the removal of hydrogen bonds that could potentially stabilize the intermediate.

[0081] This structural change may cause a shift in the folding pattern of some carbon chains during the cascade ring formation process, preventing the substrate from successfully forming a complete carbon chain. 25 Cyclization products, however, may terminate cyclization prematurely in the TC domain or undergo different oxidation pathways, thereby generating C. 10 Monoterpenoids. In addition, the S235A mutation may have altered the microenvironmental polarity of the active pocket, making oxidases or water molecules more accessible to carbocation intermediates, thus promoting the formation of oxidized monoterpenes (such as pulegone oxide).

[0082] Furthermore, the increased local flexibility of S235A may allow for higher degrees of freedom in substrate rotation or folding within the TC domain, further increasing the probability of atypical cyclization or bypass reactions, thus forming structurally similar monoterpene derivatives. Compared to the T88G mutation, S235A primarily achieves product diversification by removing side-chain hydrogen bonds and altering the polarity / flexibility of the active pocket, rather than by affecting the coupling between the PT and TC domains. This indicates that local residues in the active pocket play a decisive role in cyclization pathway selection and byproduct formation, and suggests that in semi-rational enzyme engineering, targeted product generation can be achieved through fine-tuning of key residues in the TC domain.

[0083] 5. NfSS mutant Q654A: like Figure 5 , Figure 6 As shown, GC-MS analysis results indicate that a distinct characteristic peak appears at approximately 22.0 min, with a peak area abundance of approximately 5.0 × 10⁻⁶. 6 The peak corresponds to the target disesquiterpene product Sesterfisherol; simultaneously, another significant peak was detected at approximately 11.1 min, with an abundance of approximately 4.0 × 10⁻⁶. 6 This indicates that an additional monoterpene compound was formed in the system. The molecular formula of this monoterpene is C2. 10 H16O2, with a precise MW of 168.1150 g / mol, highly matches the ion peak m / z 168.0 in the mass spectrum, suggesting that it is an oxygen-containing monoterpene.

[0084] Further mass spectrometry fragmentation analysis showed a base peak at m / z 83.0, which likely originated from C6H.11 O + or C5H9O2 + Oxygen-containing fragments are common cleavage ions of monoterpene oxides; m / z 69.0 corresponds to C5H9. + This is a typical characteristic fragment of the isoprene unit; m / z 55.0 may be C4H7. + or C3H5O + m / z 41.0 is C3H5 + These are all common cleavage ions found in terpenoid compounds. The aforementioned fragments collectively point to the isoprene skeleton structure, consistent with the basic characteristic of monoterpenes being composed of two C5 units. Furthermore, the m / z peaks at 101.0, 123.1, 138.8, and 153.0 correspond to oxygen-containing or dehydrogenated fragments formed under different cleavage pathways. The m / z peak at 138.8 may be generated by the dehydration of molecular ions, further demonstrating that this compound contains an oxygen functional group.

[0085] Based on the analysis of the molecular formula and mass spectrometry fragment characteristics, the product should be a monoterpene oxide containing two oxygen atoms, possibly belonging to linalool oxide (C42- ... 10 H 18 O), nerol oxide (C) 10 H 18 Types include O2) or cyclic monoterpene oxides. Among them, pulegone oxide (C 10 H 16 The molecular formula and characteristic fragments of O2 are highly consistent with the experimental results, especially the base peak at m / z 83.0 and characteristic ions at m / z 69.0 and m / z 41.0. Therefore, this byproduct is likely a menthol oxide compound. This product may also be a monoterpene aldehyde containing carbonyl and hydroxyl groups, which readily loses CO (m / z 28) and methyl groups upon cleavage; or it may be a cyclic monoterpene oxide, such as 1,8-cineole (C... 10 H 18 O) derivatives, which, upon cyclic cleavage, yield C6H 11 O + (m / z 83.0). The above results indicate that after expanding the substrate binding pocket, the NfSS mutant can not only catalyze the generation of the target product Sesterfisherol, but also exhibits a certain degree of product diversity, providing an important basis for further analysis of its catalytic mechanism and enzyme engineering modification.

[0086] Q654, located within the active pocket of the PT domain, typically maintains the correct positioning of the substrate isoprene chain through side-chain hydrogen bonds and steric constraint, ensuring the successful generation of the GFPP precursor and its entry into the TC domain for cascade ring formation. Replacing Gln with Ala eliminates the original polar side chain, leading to a decrease in local polarity within the active pocket and a weakening of steric constraint. This altered local environment may cause the isoprene chain to deflect or rotate prematurely within the PT domain, preventing some substrate from successfully elongating to the complete C4 domain. 25 The precursor undergoes an atypical termination reaction within the PT domain or contacts solvent molecules, initiating an oxidation-side reaction to generate C. 10 The oxidized monoterpene products. The Q654A mutation may also affect metal ions (such as Mg). 2+ The stable binding of the carbocation intermediate with the substrate pyrophosphate group reduces the stability of the carbocation intermediate, thereby increasing the chance of it being attacked by oxidases or water molecules.

[0087] Furthermore, the increased local flexibility of the PT domain may alter the kinetic pathway of substrate entry into the TC domain, prolonging the residence time of some substrates in the PT domain and thus generating oxidized monoterpenes. This differs from the T88G and S235A mutations, which primarily affect the cyclization pathway within the TC domain. The product diversification of the Q654A mutation mainly stems from the failure of the PT domain to regulate precursor chain elongation and substrate localization.

[0088] Therefore, the Q654A mutation induces the formation of oxidized monoterpenes by reducing the polarity and steric constraint of the PT active pocket, altering substrate localization and carbocation stability, revealing the central role of the PT domain in precursor chain elongation, substrate stability, and product specificity. This mechanism provides a molecular basis for the semi-rational design of multi-product enzymes and can be used to regulate the PT domain to generate diverse terpene derivatives.

[0089] comprehensive Figure 1-6 It was found that the NfSS mutant, while maintaining its ability to generate sesesterfisherol, could also catalyze the formation of various oxygen-containing monoterpenoids, such as geraniol and geranilic acid, demonstrating a significant product spectrum expansion effect. This indicates that by expanding the active pocket and optimizing key amino acid residues, the substrate specificity and catalytic pathway of NfSS can be effectively regulated, providing an important basis for the functional modification of sesquiterpene synthases and the diversified biosynthesis of terpenoid natural products.

[0090] These mutants are able to simultaneously generate Sesterfisherol (C 25 (disesquiterpenes) and C 10 Monoterpenoid compounds indicate that the catalytic pocket of NfSS may have undergone a significant conformational change after mutation. Structural remodeling allowed the enzyme to retain not only the catalytic C... 25 It possesses the ability to cyclize precursor GFPP to generate disesquiterpenes, and also has the capacity to recognize and catalyze C.10 The potential for precursor GPP to undergo cyclization or dehydration reactions. This phenomenon suggests that mutations may effectively expand the substrate binding space and reduce steric hindrance, thereby enhancing the enzyme's adaptability to substrates of different chain lengths and leading to a significant expansion of the product spectrum.

[0091] Example 3: Modification of NfSS catalytic activity I. Experimental Methods: 1. Key NfSS mutation sites identified: F637W, Q654I, and Q654R.

[0092] 2. Plasmid extraction and purification: The steps are the same as those in the plasmid extraction and purification section of Example 2.

[0093] 3. PCR primer design and amplification: according to NfSS Single-point mutation primers were designed for the mutation sites (Table 6).

[0094] Table 6. Primers for F637W, Q654I, and Q654R mutations

[0095] The PCR reaction system, PCR reaction procedure, and DpnI digestion reaction system are the same as those in Example 2, which includes PCR primer design and amplification.

[0096] 4. Plasmid transformation and positive clone screening and identification: The steps are the same as those in the plasmid transformation and positive clone screening and identification section of Example 2.

[0097] 5. Catalytic activity of mutant and GC-MS detection: The steps are the same as those for the GC-MS detection of NfSS mutant catalytic activity and Sesterfisherol yield in Example 2.

[0098] II. Results Analysis: 1. Construction and validation of NfSS F637W, Q654I, and Q654R mutant plasmids and engineered bacteria: The correct construction of the NfSS F637W, Q654I, and Q654R mutant plasmids and engineered bacteria was verified by DNA sequencing, colony PCR, and SDS-PAGE analysis.

[0099] (1) DNA Sequencing: To confirm the success of the target mutation, the constructed NfSS mutant plasmids were first sequenced using Sanger sequencing technology. The extracted plasmid DNA was quantified and purified, and then unidirectionally sequenced using specific primers. Sequencing results showed that the target mutations were successfully introduced into the NfSS F637W, Q654I, and Q654R plasmids, and there were no other unexpected mutations at the mutation sites, consistent with the expected gene sequences. Furthermore, sequence analysis verified the correctness of the inserted fragments and the target gene, ensuring the accuracy of the construction process.

[0100] (2) Colony PCR: To further confirm the correct insertion of the recombinant plasmid and the accuracy of the mutation, colony PCR was used to screen individual colonies. The target gene was amplified by PCR in each colony, and the PCR products were analyzed by agarose gel electrophoresis. The results showed that the PCR product bands of the target mutants were consistent with the expected size, and the successful construction of the NfSS F637W, Q654I, and Q654R mutants was further confirmed by comparison with the standard control.

[0101] (3) SDS-PAGE analysis: To confirm the expression of the recombinant plasmid in the engineered bacteria, SDS-PAGE analysis was performed. SDS-PAGE analysis of the whole-cell lysate showed that the NfSSPT domain mutants F637W, Q654I, and Q654R all exhibited distinct bands at the target protein (approximately 87 kDa), indicating successful expression of these mutants with good protein expression levels. Simultaneously, SDS-PAGE also revealed the purity and size of the expressed protein, further validating the correctness of the recombinant protein.

[0102] Therefore, after multiple validations using DNA sequencing, colony PCR, and SDS-PAGE, the NfSS F637W, Q654I, and Q654R mutant plasmids were successfully constructed, and their efficient expression in engineered bacteria was confirmed. These experimental results provide a reliable molecular basis for subsequent enzyme activity analysis and functional validation.

[0103] 2. Sesterfisherol yield of NfSS PT domain mutant: The catalytic product Sesterfisherol of the NfSS PT domain mutant was detected by GC-MS. Figure 7 As shown in (a), the yield of Sesterfisherol varies significantly among the mutants, providing important clues to the relationship between enzyme structure and function.

[0104] The NfSS PT domain mutants Q654I and Q654R exhibited the most significant enhancement in catalytic activity, with yields increased by approximately 63% and 59%, respectively.p <0.0001 indicates that the Q654 site plays a crucial role in substrate localization and carbocation intermediate stability. Mutations at this site may improve substrate spatial orientation in the PT domain by enhancing the stability of the active pocket conformation, optimizing the hydrophobic and hydrogen bond network, thereby increasing the efficiency of GFPP transfer to the TC domain and promoting Sesterfisherol production. The F637W mutant showed an approximately 30% increase in yield (…). p <0.0001), suggesting that aromatic residue F637 also plays an important role in intermediate stabilization and hydrophobic interaction, and its mutation can adjust the path selectivity of carbocation cascade cyclization.

[0105] Heatmap Figure 7 Figure (b) shows the relative effects of various NfSS PT domain mutants on Sesterfisherol yield. First, the red and yellow gradient regions correspond to high-yield mutants, among which Q654I, Q654R, and F637W mutants show significant yield increases, with a significantly increased peak area compared to wild-type (WT) Sesterfisherol. This indicates that mutations of these key residues optimize the PT domain's ability to regulate precursor chain elongation and substrate localization, contributing to enhanced GFPP generation and downstream cyclization efficiency. Second, the green and blue regions correspond to mutants with decreased yields, such as L512R, L512M, L512I, and F637Y, whose yields are significantly lower than WT. This suggests that these mutations may perturb the PT domain structure or substrate-binding pocket conformation, reducing precursor stability and enzyme catalytic efficiency. The horizontally arranged mutants in the heatmap (columns A–M) show the effects of different single-point and combined mutations, reflecting the sensitivity of different positions in the PT domain to catalytic efficiency. In particular, the double mutants Q654I / F637W and Q654R / F637W, while still yielding higher yields than some inefficient single mutants, did not exceed their corresponding single mutant yields, exhibiting a negative synergistic effect. This suggests that synergistic effects at key sites are crucial for structural equilibrium. Overall, the heatmap not only visually demonstrates the contribution of key residues in the PT domain to Sesterfisherol yield but also provides guidance for subsequent enzyme engineering strategies. High-yielding mutants can serve as templates for targeted modification in industrial biosynthesis, optimizing precursor utilization efficiency and product output of NfSS in large-scale fermentation systems. Meanwhile, low-yielding mutants provide a reference for further understanding the structure-function relationship of the PT domain.

[0106] In summary, the results show that F637 and Q654 are core residues in the PT domain active pocket, crucial for substrate localization, reaction pathway selection, and intermediate stability. This finding provides clear targets for semi-rational design and targeted modification of NfSS, while emphasizing the importance of overall enzyme conformation and the synergistic effect of key residues in optimizing catalytic activity, providing a theoretical basis for the efficient biosynthesis of Sesterfisherol.

[0107] Therefore, this invention conducted site-directed mutagenesis studies around key sites. The results showed that the Q654I, Q654R, and F637W mutants exhibited significantly enhanced Sesterfisherol production capacity, especially Q654I and Q654R, which were about 63% and 59% higher than the wild type, respectively. The T88G, S235A, and Q654A mutants showed obvious changes in product profiles. In addition to producing the main product Sesterfisherol, they were also able to produce geraniol, geranilic acid, and oxidized monoterpenoid byproducts. This invention realizes the transformation of NfSS from inefficient natural catalysis to efficient and controllable biosynthesis, providing a new theoretical basis and technical path for the industrial bio-manufacturing of disesquiterpenoid natural products.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A sesquiterpene synthase mutant of *Neosatoria ferri*, characterized by: The Feshiosartos disesquiterpene synthase mutant was obtained by mutating the following amino acid sequence: threonine T at position 88 to glycine G, serine S at position 235 to alanine A, glutamine Q at position 654 to alanine A, glutamine Q at position 654 to isoleucine I, glutamine Q at position 654 to arginine R, or phenylalanine F at position 637 to tryptophan W.

2. The *Neosatoria fischeri* disesquiterpene synthase mutant according to claim 1, characterized in that: The Fessiformia disesquiterpene synthase mutant uses geraniol farnesyl pyrophosphate (GFPP) as a substrate, and its product profile includes sesquiterpene fish oil alcohol (Sesterfisherol), monoterpene geraniol (Geraniol), monoterpene geranic acid (Geranilic acid), and pulegone oxide (Pulegone oxide).

3. The encoding gene of the Fexis neosatoria disesquiterpene synthase mutant according to claim 1.

4. A recombinant plasmid containing the encoding gene of the Fexitosporium disesquiterpene synthase mutant as described in claim 3.

5. The method for constructing the recombinant plasmid according to claim 4, characterized in that: Site-directed mutagenesis was performed on the amino acid sequence of sesquiterpene synthase from *Neotrichum ferri*, and the coding gene was synthesized. BamHI and HindIII restriction sites were introduced at both ends of the coding gene, and the gene was cloned into the pRSFDuet-1 vector to construct the recombinant plasmid pRSFDuet-NfSS.

6. A genetically engineered bacterium expressing the Feshiosartos disesquiterpene synthase mutant of claim 1.

7. The genetically engineered bacterium of the *Neosatoria ferrocephala* sesquiterpene synthase mutant according to claim 6, characterized in that: The genetically engineered bacteria is Escherichia coli.

8. The use of the Fexinovia disesquiterpene synthase mutant according to claim 1 in the preparation of disesquiterpene fish oil Sesterfisherol.

9. The application according to claim 8, characterized in that: Compared with the wild type, the yield of sesesterfisherol from the Feshiosartobacter disesquiterpene synthase mutant is increased by 30-63%.

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