Iridoid synthase mutants with improved specificity and activity and uses thereof
By performing site-directed amino acid mutations on the iridoid synthase NmISY2, its substrate specificity and catalytic activity for 8-oxogeraniol were improved, solving the problem of poor substrate specificity in the iridoid synthesis pathway and achieving efficient synthesis of nepetaol.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2022-10-18
- Publication Date
- 2026-07-24
Smart Images
Figure CN116716264B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a cycloene ether synthase mutant with enhanced specificity and enzyme activity and its application. Background Technology
[0002] Monoterpenoid indole alkaloids are a large class of special metabolites derived from plants. Currently, over 3000 monoterpenoid indole alkaloids have been discovered, originating from plants in families such as Apocynaceae, Rubiaceae, Loganiaceae, and Davidiaceae. Based on their structure and origin, monoterpenoid indole alkaloids can be divided into four categories: alkaloids from the genera *Conanthus*, *Epocamea*, *Alternanthera*, and quinine. Monoterpenoid indole alkaloids and their derivatives possess a wide range of pharmacological activities. For example, vincristine and vinblastine from *Catharanthus roseus* can be used as anticancer drugs, and quinine from cinchona trees can be used to treat malaria. Camptothecin from *Camptotheca acuminata* can be used to treat tumors. Large-scale preparation of monoterpenoid indole alkaloids and their derivatives is difficult to achieve through plant extraction and traditional chemical synthesis. Therefore, heterologous host synthesis of these natural products has become a research hotspot.
[0003] Nepeol is a key precursor to many monoterpene indole alkaloids. Existing methods for synthesizing nepeol using microorganisms often result in low yields. One bottleneck lies in the cycloene ether synthase pathway, where the substrate specificity of cycloene ether synthases is poor.
[0004] Iridoid synthase is a key enzyme in the cyclic ether synthase pathway. It belongs to the short-chain dehydrogenase / reductase family and can catalyze the reduction of 8-oxogeraniol to an enol intermediate, and spontaneously cyclize it to generate the key precursor nepetalol. Iridoid synthase is a substrate-mixed enzyme that can reduce geraniol, an oxidation product of the precursor geraniol, to citronellol. This causes the metabolic flux in the cyclic terpenoid synthesis pathway to deviate from the target pathway, which is not conducive to the synthesis of nepetalactone and its subsequent products. ([1] Geuflores F. et al. Identification and characterization of the iridoid synthase involved in oleuropein biosynthesis in olive,fruits. 2015. [2] Geu-Flores, F., Sherdavault, N., Courtavault, V. et al. An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492, 138–142 (2012). [3] Sherden NH, Lichman B, Caputi L, et al. Identification of iridoid synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry. Phytochemistry. 2018; 145: 48-56. [4] Wu Shiwen, Yang Mengquan, Xiao Youli. Research progress in the synthetic biology of monoterpenoid indole alkaloids [J]. Organic Chemistry, 2018, 38(9): 16. Figure 1 The chemical equations for the catalytic conversion of geranialdehyde to citronellol by iridoid synthase and the catalytic conversion of 8-oxogeranialdehyde to nepetaol.
[0005] Therefore, obtaining highly specific and highly active cycloenol synthases using genetic engineering techniques will help overcome the bottlenecks in the current cycloenol synthesis pathway and promote the biosynthesis and application of monoterpenoid indole alkaloids. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides a mutant of iridoid synthase with enhanced substrate specificity. The mutation involves altering the internal amino acids of iridoid synthase (NmISY2) derived from Nepeta mussinii, as shown in SEQ ID NO.1, thereby improving the substrate specificity and catalytic activity of iridoid synthase for 8-oxogermanylase.
[0007] This invention first provides a mutant of iridoid synthase with enhanced specificity and enzyme activity, obtained by mutating the wild-type iridoid synthase NmISY2 derived from Nepeta mussinii. The amino acid sequence of the wild-type iridoid synthase NmISY2 is shown in SEQ ID. As shown in NO.1, the mutation site contains one of the following mutations: W109Y, W109S, V157D, M217D, M218D, K343A, K343D, K343M, W345D, W109S / K343D, W109S / W345D, W109Y / K343M, W109Y / W345D, K343M / W345D, W109S / V157D / M217D, W109N / K343A / W345D, W109Y / V157D / W345D, W109Y / M217D / W345D, W109Y / K343M / W345D, or W109Y / M217D / K343M / W345D.
[0008] The present invention further provides a gene encoding the iridoid synthase mutant.
[0009] This invention also provides an expression vector containing the gene. The expression vector includes a plasmid backbone and the gene sequence. Preferably, the expression vector is a pET-28a plasmid containing the gene. There are no specific limitations on the plasmid used to express the iridoid synthase mutant and the strain used for expression in this application, as long as the iridoid synthase mutant can be correctly expressed.
[0010] The present invention also provides genetically engineered bacteria containing the aforementioned gene.
[0011] This invention further provides the application of the iridoid synthase mutant in the preparation of nepetaol. In application, the purified iridoid synthase mutant is added to the system as a catalyst.
[0012] The present invention also provides the application of the gene in the preparation of nepetaol.
[0013] The present invention further provides the application of the genetically engineered bacteria in the preparation of nepetaol.
[0014] The present invention also provides a method for preparing nepetalol, using 8-oxogeranium as a substrate, NADPH as a coenzyme, and utilizing the iridoid synthase mutant or the genetically engineered bacteria as a catalyst to catalytically reduce and generate nepetalol.
[0015] The beneficial effects of this invention are:
[0016] This invention utilizes site-directed mutagenesis to mutate the amino acids in the substrate-binding pocket of cycloenol synthase, altering the interaction between the enzyme molecule and its substrate. This alters the substrate-binding mechanism, thereby improving the substrate specificity and activity of the cycloenol synthase. The mutant of this invention exhibits high catalytic activity against 8-oxogeraniol, but shows a significant decrease in activity against geraniol. The mutant of this invention achieves a maximum activity ratio of 293 times for 8-oxogeraniol and geraniol, representing a substantial improvement over the parental cycloenol synthase. This mutant can be used for the heterologous synthesis and in vitro synthesis of cycloenol compounds and their derivatives, showing promising application prospects. Attached Figure Description
[0017] Figure 1 The chemical equations for the catalytic conversion of geranialdehyde to citronellol by iridoid synthase and the catalytic conversion of 8-oxogeranialdehyde to nepetaol.
[0018] Figure 2 These are gas chromatography-mass spectra of different samples. Detailed Implementation
[0019] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.
[0020] Reagents used in upstream genetic engineering operations: The restriction endonucleases, Primer STAR DNA polymerase, DNA ligase, and recombinase used in the embodiments of this invention were all purchased from TaKaRa; the genome extraction kit, plasmid extraction kit, and DNA recovery and purification kit were from Axygen; E. coil BL21(DE3), plasmids, etc., were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and gene sequencing were performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. Refer to the product instructions for the usage of the above reagents.
[0021] Reagents used in downstream catalytic processes: Geranialdehyde was purchased from Jiangsu Aikon Biomedical R&D Co., Ltd., 8-O-geranialdehyde was purchased from TRC Company, NADPH was purchased from Bangtai Bioengineering Co., Ltd., and other commonly used reagents were purchased from commercially available analytical grade reagents.
[0022] Example 1: Cloning of the wild-type iridoid synthase gene
[0023] The amino acid sequence encoding iridoid synthase (derived from *Nepeta mussinii*) was obtained by inputting ASM62112.1 into the National Coalition Building Institute (NCBI) database. Based on the codon bias of *E. coli*, the amino acid sequence (as shown in SEQ ID NO.1) was converted into a nucleotide sequence, as shown in SEQ ID NO.2. The nucleotide sequence was then chemically synthesized (by General Biotechnology (Anhui) Co., Ltd.) and integrated into the expression vector pET-28a at a seamless cloning site—XhoI. Finally, the constructed plasmid was introduced into *E. coil* BL21(DE3) to construct a wild-type engineered iridoid synthase strain.
[0024] Example 2: Construction of a cyclopentadiene synthase mutant
[0025] 1. Activation of engineered bacteria and plasmid extraction
[0026] All engineered bacteria (obtained in Example 1) were activated and cultured using LB medium with the following formulation: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The culture was then sterilized at 121°C for 20 min and set aside for later use. The solid culture medium was LB medium with 2% (w / w) agar added.
[0027] The preserved engineered bacteria glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored at -80°C for long-term storage.
[0028] 2. Site-directed gene mutation
[0029] Gene mutations are performed using whole plasmid PCR. When only a small-scale mutation is needed, the mutation can be designed into the upstream and downstream primers, and PCR can be performed using the plasmid as a template to obtain the target plasmid.
[0030] Table 1 PCR amplification system
[0031]
[0032]
[0033] PCR amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55-58℃ annealing for 15 s (adjust according to primer Tm value), 72℃ extension for 75 s (adjust according to gene length, 10 s / 1 kb), for a total of 30 cycles; 72℃ extension for 10 min; store at 4℃.
[0034] After PCR amplification, the amplification products were detected by 0.9% (w / v) agarose gel electrophoresis. The results showed that the amplification products were a single band. The amplification products were purified and recovered using a DNA recovery kit. The specific steps were followed according to the instructions of the purification kit.
[0035] The upstream and downstream primers for the 345th tryptophan mutation to aspartic acid in iridoid synthase are:
[0036] Upstream primer: 5'-GTGGGTAAATGGgatTTTAGTGATACCATTCTGTGGAATGA-3';
[0037] Downstream primer: 5'-AAatcCCATTTACCCACATCTTCCAGTTTGGT-3';
[0038] Other mutation point primers were designed according to this principle.
[0039] 3. Construction of mutant engineered bacteria
[0040] The purified gene fragment was digested with DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into E. coil BL21(DE3) competent cells, plated, and single colonies were picked and cultured in LB liquid. Positive transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, sterile glycerol was added to a final concentration of 20% (v / v), labeled, and stored at -80℃ for later use.
[0041] Example 3: Expression and purification of iridoid synthase mutant
[0042] LB liquid culture medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, sterilized at 121℃ for 20 min, ready for use.
[0043] 1. Preparation of crude enzyme solution
[0044] After activation by streaking in a petri dish, engineered bacteria containing the iridoid synthase gene were inoculated as single colonies into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 12 h. Then, 2% of the inoculum was transferred to 50 mL of fresh LB broth containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD (digesterone) was reached.600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM and induce culture at 18℃ for 16-18 hours. After the culture is completed, centrifuge the culture medium at 4000 rpm for 10 minutes, discard the supernatant, collect the bacterial cells, and store them in an ultra-low temperature freezer at -80℃ for later use.
[0045] Centrifuge the collected fermentation broth at 4000 rpm for 15-20 min, discard the supernatant and retain the lower layer of cells, and wash the cells 2-3 times with an appropriate amount of buffer. Resuspend the washed cells in MOPS buffer and disrupt the cells using an ultrasonic cell disruptor. The conditions for ultrasonic cell disruption are: disruption power of 30% of the total power, disruption time of 2 seconds, interval of 3 seconds, and a cycle count of 300 times. The temperature for ultrasonic cell disruption is strictly controlled at 4℃. Centrifuge the disrupted cell culture at 12000 rpm for 15 min, and the resulting supernatant is the crude enzyme solution of iridoid synthase.
[0046] 2. Purification of iridoid synthase
[0047] The expressed iridoid synthase has a 6×His fusion tag at the C-terminus. Since the imidazole group of histidine can chelate with Ni ions, the protein was purified using a Ni-NTA protein purification column.
[0048] The specific operating steps are as follows:
[0049] Pretreatment of protein samples and Ni-NTA protein purification column: The obtained crude iridoid synthase solution was filtered through a 0.22 μm filter membrane for further impurity removal. The Ni-NTA protein purification column was rinsed with 10 column volumes of ultrapure water to ensure that 20% (v / v) ethanol was completely removed from the column. The Ni-NTA protein purification column was then equilibrated with 10 column volumes of 50 mM imidazole solution.
[0050] Protein purification and elution: The filtered nitrile hydrolase protein sample was loaded twice. The Ni-NTA affinity column was washed with 10 column volumes of 50 mM imidazole solution to remove unbound contaminating proteins. The Ni-NTA affinity column was then washed with 10 column volumes of 300 mM imidazole to elute the target protein. The eluted target protein was collected and stored on ice.
[0051] Protein ultrafiltration desalting: The collected target protein was concentrated by centrifugation at 4000 rpm for 10 min at 4°C using a Millipore ultrafiltration tube with a molecular weight cutoff of 10 kDa. After concentration, the protein concentration was determined by the Bradford method and immediately used for enzyme activity assay.
[0052] Example 4: Preparation of Nepetaol from Iridoid Synthase and its Mutants
[0053] Purified enzymes of wild-type iridoid synthase and its mutants were obtained according to the method in Example 3. A 1 mL system was established with a substrate concentration of 100 μM and an NADPH concentration of 200 μM. The reaction temperature and rotation speed were controlled using a metal bath. After incubating the reaction solution at 30°C and 650 rpm for 15 minutes, the samples were identified using gas chromatography-mass spectrometry (GC-MS).
[0054] Identification method: A programmed temperature ramp was used. After holding at 70°C for 1 minute, the column temperature was increased to 180°C at a rate of 30°C / min and held for 1 minute, then increased to 265°C at a rate of 50°C / min and held for 6 minutes. The ion source interface temperature was 230°C, and the interface temperature was 270°C, with a solvent delay allowed for 3 minutes. Selected ion scanning mode was used, such as... Figure 2 As shown, nepetaol ( Figure 2 -a) The quantitative ion concentration was 168 m / z, and the qualitative ion concentrations were 150 m / z and 135 m / z, with a peak elution time of approximately 9.2 minutes; 8-oxogeranium ( Figure 2 The quantitative ion concentration (QI) for -b) was 148 m / z, the qualitative ion concentration (QI) was 137 m / z, and the elution time was approximately 11.8 minutes.
[0055] Example 5: Determination of specific enzyme activity and specificity of iridoid synthase and its mutants
[0056] The purified enzyme was obtained according to the method in Example 3. The enzyme solution was diluted to 0.001 mg / mL or 0.05 mg / mL with MOPS buffer (pH 7.0) and used for reaction with 8-oxogeraniol or geraniol, respectively. A 1 mM substrate stock solution and a 2 mM coenzyme stock solution (NADPH) were prepared using MOPS buffer. The substrate and enzyme solution were incubated at 30°C for 10 minutes each. 20 μL of NADPH and 20 μL of substrate were added to the microplate, followed by 160 μL of enzyme solution. The mixture was then pipetted and the enzyme activity was measured using a microplate reader. The microplate reader was used in time-scan mode, with the temperature set to 30°C. The absorbance of the reaction solution was measured at 340 nm, with readings taken every 8 seconds. The obtained data were linearly fitted, and the slope of the obtained curve was divided by the slope of the coenzyme standard curve, and the spontaneous decay rate of the coenzyme was subtracted to obtain the measured enzyme activity. The enzyme specificity was represented by the ratio of the specific enzyme activities to 8-oxogeraniol and geraniol, Q. Data are shown in Table 2.
[0057] Table 2. Specific enzyme activities and Q values of iridoid synthases and their mutants catalyzing geraniol or 8-oxogeraniol.
[0058]
[0059]
[0060] Compared with the wild-type iridoid synthase NmISY2, the iridoid synthase mutants showed decreased specific enzyme activity for geraniol and increased specific enzyme activity for 8-oxogeraniol. Among them, W109Y / M217D / K343M / W345D showed the best substrate specificity, with a specific enzyme activity of 48.6 U / mg for geraniol, which was about 84% lower than that of the parent. The specific enzyme activity of this mutant for 8-oxogeraniol was 14238.1 U / mg, which was 5.5 times that of the parent. The ratio of the specific enzyme activities of geraniol and 8-oxogeraniol catalyzed by this mutant was increased by nearly 35 times.
[0061] The above data show that the substrate specificity and enzyme activity of the mutant of this invention have been improved, and it has good application prospects.
Claims
1. A mutant of cycloenzyme synthase with enhanced specificity and enzyme activity, characterized in that, Derived from the catnip of Mu ( Nepetamussinii Wild-type iridoid synthase Nm ISY2 mutant, wild-type iridoid synthase Nm The amino acid sequence of ISY2 is shown in SEQ ID NO.
1. The mutation site is one of the following mutations: W109Y, W109S, V157D, M217D, M218D, K343A, K343D, K343M, W345D, W109S / K343D, W109S / W345D, W109Y / K343M, W109Y / W345D, K343M / W345D, W109S / V157D / M217D, W109Y / V157D / W345D, W109Y / M217D / W345D, W109Y / K343M / W345D, or W109Y / M217D / K343M / W345D.
2. The gene encoding the iridoid synthase mutant of claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector according to claim 3, characterized in that, For inserting a pET-28a plasmid containing the gene as described in claim 2.
5. Genetically engineered bacteria containing the gene of claim 2.
6. The application of the iridoid synthase mutant of claim 1 in the preparation of nepetaol.
7. The use of the gene described in claim 2 in the preparation of nepetaol.
8. The use of the genetically engineered bacteria according to claim 5 in the preparation of nepetaol.
9. A method for preparing nepetaol, characterized in that, Using 8-oxogeranium as a substrate and NADPH as a coenzyme, and employing the iridoid synthase mutant of claim 1 or the genetically engineered bacteria of claim 5 as a catalyst, nepetaol is catalytically reduced to form nepetaol.