Cloning method and application of geraniol synthetase gene

Abstract

The invention belongs to but is not limited to the technical field of molecular biology, and particularly relates to a cloning method and application of a geraniol synthetase gene. According to the strain XY04, saccharomyces cerevisiae is used as a starting strain, overexpression tHMGR, UPC2-1, Erg20F96W-N127W and CoTPS1 are carried out, the tHMGR and UPC2-1 genes are integrated to a saccharomyces cerevisiae chromosome TY3 site, the Erg20F96W-N127W is integrated to an ROX1 site, and the CoTPS1 gene is integrated to a saccharomyces cerevisiae chromosome TY2 site.

Description

Technical Field

[0001] The invention belongs to but is not limited to the technical field of molecular biology, and in particular relates to a cloning method of a geraniol synthase gene and an application thereof. Background Art

[0002] Geraniol, also known as geraniol, is a naturally occurring monoterpenoid unsaturated alcohol widely distributed in the essential oils of various plants such as rose (Rosa damascena), geranium (Pelargonium graveolens), and citronella (Cymbopogon spp.). As an acyclic monoterpene alcohol, geraniol has a unique sweet and mellow floral fragrance and is a key component for blending rose, citrus and lavender fragrances. It has been widely used in high-end perfumes, skin care products and many spices. Modern medical research shows that geraniol has rich pharmacological activities, covering multiple functions such as anti-tumor, anti-inflammatory, antioxidant, antibacterial, organ protection and metabolic regulation. As an important member of the terpene family, geraniol has shown significant application value in spices, medicine, agriculture and food industries. With the growing demand for natural product development, the potential of geraniol as a substitute for synthetic spices and a multifunctional bioactive molecule has attracted much attention. In recent years, it has become a hot spot in natural product development and green biotechnology research. Although geraniol can be obtained by direct separation and extraction from plants, as well as chemical synthesis, the extraction and separation of geraniol is inefficient due to its extremely low content in plants, while chemical synthesis faces multiple challenges such as complex byproducts and easy oxidation of double bonds. Therefore, using synthetic biology to prepare it through microbial cell factories has become an effective way to solve the production problem of this high-value ingredient.

[0003] Celastrus orbiculatus is a plant of the genus Celastrus in the family Celastraceae. As a traditional Chinese medicine with extensive medicinal value, it has a long history of use in my country. It has the effects of detoxification and swelling, wind and dampness removal, menstruation and pain relief, blood circulation and blood stasis, tranquilization, anti-inflammatory, and strengthening bones and muscles. Modern pharmacological studies have shown that Celastrus orbiculatus has a variety of biological activities such as anti-tumor, anti-multidrug resistance, anti-atherosclerosis, anti-inflammatory and anti-rheumatic, anti-viral, and antioxidant, especially for a variety of cancers (such as liver cancer, gastric cancer, cervical cancer, esophageal cancer, and colon cancer). Terpenoid compounds are an important category of plant secondary metabolites, and are also the material basis for the efficacy of Celastrus orbiculatus and the key to the formation of authentic medicinal materials. At present, a variety of terpenoid compounds have been identified and separated from Celastrus orbiculatus, among which monoterpenoid components have attracted much attention due to their significant biological activities. However, no monoterpene synthase has been reported in Celastrus orbiculatus so far.

[0004] In view of the above analysis, the technical problem that urgently needs to be solved in the prior art is: there is currently a lack of research on monoterpene synthase in Celastrus orbiculatus. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides

[0006] The present invention is achieved by providing a geraniol synthase gene, characterized in that its nucleotide sequence is shown in the sequence list SEQ ID NO.1; the amino acid residue sequence of the gene-encoded protein is shown in the sequence list SEQ ID NO.2;

[0007] Another object of the present invention is to provide a method for cloning the geraniol synthase gene CoTPS1, and the specific steps are as follows:

[0008] (1) Extracting total RNA from Celastrus orcidudata and reversely transcribing it into cDNA;

[0009] (2) Using cDNA as a template, specific primers were designed to amplify the geraniol synthase gene CoTPS1 by PCR;

[0010] (3) Recovering and purifying the PCR product in step (2) by agarose gel electrophoresis and an agarose gel DNA recovery kit to obtain the geraniol synthase gene CoTPS1 fragment.

[0011] Furthermore, PCR amplification was performed using 2x Phanta Max Master Mix (P515-03, Nanjing Novozymes Biotech Co., Ltd.) high-fidelity DNA polymerase.

[0012] Furthermore, the PCR amplification system was 10 μL of 2x Phanta Max Master Mix, 0.5 μL of each forward and reverse primer, 8 μL of ddH2O, 1 μL of template, and a total reaction system of 20 μL.

[0013] The PCR amplification reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 1 min, 32 cycles; 72°C for 5 min.

[0014] Another object of the present invention is to provide an application method of the geraniol synthase gene CoTPS1 to construct an engineered bacterium for synthesizing geraniol, wherein the strain is based on Saccharomyces cerevisiae as a starting strain, overexpressing tHMGR, UPC2-1, Erg20 F96W-N127W and CoTPS1; wherein:

[0015] (1) tHMGR and UPC2-1 genes were integrated into the TY3 locus of the Saccharomyces cerevisiae chromosome;

[0016] (2) Erg20 F96W-N127W Integrated into the ROX1 locus;

[0017] (3) The CoTPS1 gene was integrated into the TY2 locus of the Saccharomyces cerevisiae chromosome;

[0018] Furthermore, the brewer's yeast is brewer's yeast BY4741.

[0019] Further, the method for constructing an engineered strain of Saccharomyces cerevisiae for synthesizing geraniol comprises the following steps:

[0020] (1) Use overlapping PCR to construct the following modules:

[0021] (a) GAL1p, tHMGR and ADH1t were connected to construct the gene expression module GAL1p-tHMGR-ADH1t, named module I;

[0022] (b) GAL10p, UPC2-1 and CYC1t were connected to construct the gene expression module GAL10p-UPC2-1-CYC1t, which was named module II;

[0023] (c) connecting module II and module III to construct the gene expression module GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t, named module III;

[0024] (d) GAL10p, Erg20 F96W-N127W Connected with CYC1t to construct the gene expression module GAL10p-Erg20 F96W -N127W -CYC1t, named module IV;

[0025] (e) Template IV and HIS selection marker are connected to construct the gene expression module HIS-V, which is named module V;

[0026] (f) GAL1p, CoTPS1 and ADH1t were connected to construct the gene expression module GAL1p-CoTPS1-ADH1t, named module VI;

[0027] (2) Construction of strain XY01

[0028] Insert module III into the SfaSI restriction site of vector pCfB2875 to obtain the integration expression vector pCfB2875-III; then cut the integration expression vector pCfB2875-III with restriction endonuclease NotΙ to obtain DNA integration fragment S1; then integrate DNA integration fragment S1 into the TY3 site of the chromosome of yeast engineering bacteria to obtain strain XY01;

[0029] (3) Construction of strain XY02

[0030] The pSH65 vector was transformed into strain XY01, and then screened to obtain strain XY02;

[0031] (4) Construction of strain XY03

[0032] Transform the module V into the yeast engineering strain XY02 to obtain the strain XY03;

[0033] (5) Construction of strain XY04

[0034] Inserting module VI into the SfaSI restriction site of vector pCfB2797 to obtain the integrated expression vector pCfB2797-VI; inserting module IX into the NheI restriction site of vector pCfB2797-VI to obtain the integrated expression vector pCfB2797-VI-IX; then cutting the integrated expression vector pCfB2797-VI-IX with restriction endonuclease NotΙ to obtain DNA integrated fragment S2; then integrating DNA integrated fragment S2 into the TY2 site of chromosome of strain TX04 to obtain strain XY04, and obtaining the high-yield santalol-producing Saccharomyces cerevisiae engineered strain;

[0035] Further, the tHMGR, UPC2-1 and Erg20 described in step (1) F96W-N127W The gene can be cloned from the genome of Saccharomyces cerevisiae BY4741, and its nucleotide sequence is shown in SEQ ID NOs. 3-5.

[0036] The GAL1p and GAL10p described in step (1) are promoter sequences, which can be cloned from the genome of Saccharomyces cerevisiae BY4741, and their nucleotide sequences are shown in SEQ ID NOs. 7 to 8, respectively.

[0037] The ADH1t and CYC1t described in step (1) are terminator sequences, which can be cloned from the genome of Saccharomyces cerevisiae BY4741, and their nucleotide sequences are shown in SEQ ID NOs. 9 to 10, respectively.

[0038] The nucleotide sequence of the screening marker HIS described in step (1) is shown in SEQ ID NO.11.

[0039] Another object of the present invention is to provide an application of the geraniol synthase gene CoTPS1 in the construction of cerevisiae engineering bacteria of geraniol and other terpenoid compounds with geraniol as a substrate.

[0040] Furthermore, the engineered yeast of brewer's yeast for synthesizing geraniol is activated and inoculated into a fermentation medium for fermentation culture to obtain geraniol.

[0041] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0042] The present invention identifies and clones a new geraniol synthase gene CoTPS1 for the first time. The gene has high catalytic efficiency and can significantly improve the synthesis rate of geraniol. Geraniol is an important monoterpene compound and is widely used in food, flavors and fragrances, medicine and other industries. However, the existing chemical synthesis method has the problems of high cost and serious pollution, while the natural plant extraction method is limited by the constraints of low yield and complex extraction process. Therefore, the present invention screens and identifies the efficient geraniol synthase gene CoTPS1 from a specific species, and optimizes its heterologous expression in Saccharomyces cerevisiae, so that the microbial cell factory can efficiently and sustainably produce geraniol, providing a new technical solution for industrial-scale production.

[0043] The technical solution of the present invention breaks through the bottleneck of efficient microbial synthesis of geraniol and solves the technical challenges that have long been difficult to overcome in the field of synthetic biology. The geraniol synthase encoded by the CoTPS1 gene has higher substrate affinity and catalytic conversion efficiency in Saccharomyces cerevisiae. Compared with the reported synthase, it shows more excellent enzyme activity and product selectivity, effectively reduces the generation of by-products, and increases the biosynthetic yield of geraniol. In addition, the present invention combines metabolic engineering strategies to optimize the precursor synthesis pathway, enzyme expression level and cell metabolic flow of the host bacteria, so that the recombinant strain can use renewable carbon sources (such as glucose) as substrates to achieve efficient fermentation production of geraniol, breaking through the yield bottleneck of natural plant sources, and significantly reducing production costs, providing a more competitive microbial manufacturing platform for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a GC-MS detection result diagram of the expression of the cloned geraniol synthase gene CoTPS1 in yeast provided in an embodiment of the present invention.

[0045] Figure 2 It is a mass spectrum of the GC-MS result of expressing the cloned geraniol synthase gene CoTPS1 in yeast provided in an embodiment of the present invention.

[0046] Figure 3 The diagram is a schematic diagram of the construction of an engineering strain of Saccharomyces cerevisiae for synthesizing geraniol provided in an embodiment of the present invention.

[0047] Figure 4 It is a schematic diagram of the yield of geraniol in the engineered strain of Saccharomyces cerevisiae for synthesizing geraniol provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] The reagents and culture medium involved in the embodiments of the present invention are as follows:

[0050] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L or galactose 18 g / L + glucose 2 g / L (add 20 g / L agar powder when preparing solid YPD medium).

[0051] SD-HIS medium: SD powder 6.7 g / L, HIS (methionine) deficient amino acid (100X) 10 mL / L, glucose 20 g / L (add 20 g / L agar powder when preparing solid culture medium).

[0052] HIS / MET / LEU / URA four-deficient amino acid mother solution (100X): arginine 0.12g, aspartic acid 0.6g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, threonine 1.2g, tryptophan 0.24g, tyrosine 0.18g, valine 0.9g, dilute to 57mL with distilled water, and any amino acid can be omitted as needed to prepare the deficient amino acid mother solution (100X) (methionine (MET) 0.12g, uracil (URA) 0.12g, leucine (LEU) 0.36g, histidine (HIS) 0.12g). The above raw materials were purchased from Sigma-Aldrich.

[0053] Amino acid stock solution (lacking HIS / MET / LEU / URA) (100X): 0.4 g adenine sulfate, 0.2 g arginine, 1 g aspartic acid, 1 g glutamic acid, 0.3 g lysine, 0.5 g phenylalanine, 3.75 g serine, 2 g threonine, 0.4 g tryptophan, 0.3 g tyrosine, 1.5 g valine, dilute to 100 mL with ultrapure water. The above raw materials were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0054] Weigh 0.2 g of methionine (MET), 0.2 g of uracil (URA), 0.6 g of leucine (LEU), and 0.2 g of histidine (HIS), and dilute to 100 mL with ultrapure water to prepare methionine (MET) mother solution (100X), uracil (URA) mother solution (100X), leucine (LEU), and histidine (HIS) mother solution (100X).

[0055] If necessary, any one of HIS / MET / LEU / URA can be added to the amino acid stock solution to prepare a defective amino acid stock solution (100X).

[0056] Example 1 Cloning of the geraniol synthase gene CoTPS1

[0057] The cloning of the linoleic acid synthase gene FAD2 of the present invention is obtained according to the following method;

[0058] 1. Extract total RNA from Celastrus orbiculatus using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441, Tiangen Biochemical Technology (Beijing) Co., Ltd.):

[0059] (1) Lysis treatment: The Celastrus orbiculatus plant tissue was quickly frozen in liquid nitrogen and ground into powder in a mortar. The powder was transferred to a 1.5 mL centrifuge tube, 500 μL of lysis solution SL was added, and mixed.

[0060] (2) Centrifuge at 12,000 rpm for 2 min.

[0061] (3) Transfer the supernatant to the filtration column CS (the filtration column CS is placed in the collection tube), centrifuge at 12,000 rpm for 2 min, and carefully pipette the supernatant in the collection tube into a new RNase-Free centrifuge tube, trying to avoid contact between the pipette tip and the cell debris precipitate in the collection tube.

[0062] (4) Slowly add 0.4 times the volume of the supernatant anhydrous ethanol and mix well (precipitate may appear at this time). Transfer the resulting solution and precipitate into the adsorption column CR3. Centrifuge at 12,000 rpm for 15 seconds. Pour out the waste liquid in the collection tube and put the adsorption column CR3 back into the collection tube.

[0063] (5) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0064] (6) Preparation of DNase I working solution: Take 10 μL of DNase I storage solution and place it in a new RNase-Free centrifuge tube, add 70RDD buffer, and mix gently.

[0065] (7) Add 80 μL of DNase I working solution to the center of adsorption column CR3 and leave at room temperature for 15 min.

[0066] (8) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0067] (9) Add 500 μL of rinse solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), centrifuge at 12,000 rpm for 15 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0068] (10) Repeat step 9.

[0069] (11) Centrifuge at 12,000 rpm for 2 min. Place the adsorption column CR3 in a new RNase-Free centrifuge tube. Add 30-50 μL RNase-Free ddH2O to the middle of the adsorption membrane. Leave at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min to obtain the RNA solution.

[0070] 2. Reverse transcription into cDNA using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (R212-01, Nanjing Novogene Biotech Co., Ltd.), the specific steps are as follows:

[0071] (1) Denaturation of RNA template: Add 4-6 μL total RNA and 2 μL Oligo(dT)23VN (50 μM) to a 200 μL RNase-free centrifuge tube, add RNase-free ddH2O to a total volume of 12 μL, heat at 65°C for 5 min, and quickly place on ice for 2 min.

[0072] (2) Removal of genomic DNA: Add 4 μL of 4x gDNA wiper mix to the mixture in the previous step and mix by gently pipetting. Incubate at 42°C for 2 min.

[0073] (3) Synthesis of the first-strand cDNA: Add 2 μL 10 x RT Mix and 2 μL HiScriptII Enzyme Mix to the mixture in the previous step, mix by gently pipetting, react at 55°C for 5 min and then at 85°C for 2 min. The product obtained after the reaction is Celastrus orbiculatus cDNA.

[0074] 3. Using the cDNA obtained above as a template, clone the geraniol synthase gene CoTPS1. The specific steps are as follows:

[0075] (1) PCR reaction system: 2x Phanta Max Master Mix 10 μL, forward and reverse primers (CoTPS1-F, CoTPS1-R) 0.5 μL each, ddH2O 8 μL, template 1 μL, total reaction system 20 μL.

[0076] The PCR amplification reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 1 min, 32 cycles; 72°C for 5 min.

[0077] After the PCR reaction is completed, 0.8-1% agarose gel is prepared, and the PCR product is added to 2 μL 10x DNA loading buffer for agarose gel electrophoresis. Trans5K DNA Marker is used to analyze the DNA bands in agarose gel electrophoresis. After confirming that the band size is correct, the target band is cut out and gel recovery is performed using an agarose gel DNA recovery kit (DP209-03, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The obtained fragment is the geraniol synthase gene CoTPS1. The specific operation process is shown in the instruction manual.

[0078] 4. Connect the DNA fragment obtained above to the pEASY-Blunt vector. The specific steps are as follows:

[0079] (1) The DNA fragment was connected to the pEASY-Blunt vector (CB111-01, Beijing Quanshijin Biotechnology Co., Ltd.) and then transformed into competent Escherichia coli DH5α. 3.5 μL of the recombinant system was added to 35 μL of competent Escherichia coli DH5α, incubated on ice for 25 min, heat-shocked for 1 min, and then incubated on ice for 2 min. 200 μL of LB medium was added and cultured at 37°C with shaking at 220 rpm for 40 min. After plating, the culture was cultured overnight for 14 to 16 h.

[0080] (2) After culturing at 37°C for 14-16 h, single clones were selected for colony PCR.

[0081] PCR reaction system: 2x M5 HiPer Taq HiFi PCR mix 5 μL, forward and reverse primers CoTPS1-F and CoTPS1-R (Table 1) 0.3 μL each, ddH2O 4.4 μL.

[0082] PCR amplification conditions were: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.

[0083] (3) After the PCR reaction was completed, agarose gel electrophoresis was performed, and the positive clone strain was picked and inoculated into 4-6 mL of LB medium containing carbenicillin for overnight culture. The plasmid was extracted using a plasmid extraction kit (DP103-03, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The specific operation process is shown in the instruction manual.

[0084] Example 2 Cloning of yeast endogenous genes, promoters and terminators

[0085] 1. Extraction of yeast genome:

[0086] The genomic DNA of Saccharomyces cerevisiae was extracted using a DNA extraction kit (DC104-01, Nanjing Novozyme Biotechnology Co., Ltd.). The specific operation process is as follows:

[0087] (1) Take 10 mL of overnight cultured yeast and centrifuge at 12,000 rpm for 1 min. Collect the yeast cells and add liquid nitrogen to grind them into powder. Transfer the powder to a 1.5 ml centrifuge tube.

[0088] (2) Immediately add 400 μL Buffer A1 and 4 μL RNase A to the above 1.5 ml centrifuge tube and vortex.

[0089] (3) Incubate in a 65°C water bath for 10 min. Invert the tube 2 to 3 times to mix the sample.

[0090] (4) Add 130 μL of Buffer A2 to the mixture, mix thoroughly, place on ice for 5 min, centrifuge at 14,000 rpm for 10 min, and carefully pipette the supernatant into a new 1.5 mL centrifuge tube.

[0091] (5) Add 1.5 times the volume of supernatant Buffer A3 and mix immediately by pipetting.

[0092] (6) Transfer the mixture obtained in the previous step to FastPure gDNA Columns IV, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0093] (7) Add 600 μl of Buffer AW, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0094] (8) Repeat step (7).

[0095] (9) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min. After centrifugation, open the cover and leave the empty column for 5 min to allow the remaining ethanol to evaporate completely.

[0096] (10) Place the adsorption column in a new 1.5 ml centrifuge tube. Add 50 μl of Elution Buffer preheated to 65°C to the center of the membrane of the adsorption column, leave at room temperature for 3 min, and centrifuge at 12,000 rpm for 1 min. After centrifugation, discard the adsorption column and store the obtained yeast genomic DNA at -20°C.

[0097] 2. Cloning of endogenous yeast genes and expression elements:

[0098] (1) Using the yeast genome obtained above as a template, clone the following genes, constitutive promoters and terminators respectively (amplification primers are shown in Table 1):

[0099] Gene tHMGR (primers tHMGR-F, tHMGR-R), gene UPC2 (primers UPC2-F, UPC2-R), gene Erg20 (primers Erg20-F, Erg20-R), promoter GAL1p (primers GAL1p-F, GAL1p-R), promoter GAL10p (primers GAL10p-F, GAL10p-R), terminator ADH1t (primers ADH1t-F, ADH1t-R), terminator CYC1t (primers CYC1t-F, CYC1t-R).

[0100] PCR reaction system: 2x Phanta Max Master Mix 10μL, forward and reverse primers 0.5μL each, ddH2O 8μl, template 1μL, total reaction system 20μL.

[0101] The PCR amplification reaction conditions were as follows: 95°C for 5 min; 95°C for 15 s, 58°C for 15 s, 72°C for 30 s for 2 min, 32 cycles; 72°C for 5 min.

[0102] After the PCR reaction, 0.8-1% agarose gel was prepared, and the PCR product was added to 2 μL 10x DNA loading buffer for agarose gel electrophoresis. Trans5K DNA Marker was used to analyze the DNA bands in agarose gel electrophoresis. After confirming that the band size was correct, the target band was cut out and gel recovery was performed using an agarose gel DNA recovery kit (DP209-03, Tiangen Biochemical Technology (Beijing) Co., Ltd.). The specific operation process is shown in the instruction manual.

[0103] (2) The DNA fragment obtained above was connected to the pEASY-Blunt vector, and then transformed into competent E. coli DH5α, cultured at 37°C overnight, and single clones were selected for colony PCR. The specific operation was the same as step 4 of Example 1.

[0104] (3) After the PCR reaction is completed, agarose gel electrophoresis is performed and the positive clone strains are selected for sequencing.

[0105] Table 1 Primer sequences used for cloning genes and expression elements in the present invention

[0106]

[0107]

[0108] Example 3: Construction of each expression module

[0109] S1. Construction of gene expression modules I to VI using overlapping PCR

[0110] (1) Each expression module was constructed using overlapping PCR technology. The connected fragments in the module were obtained by PCR cloning. The primers of adjacent fragments were designed to overlap by 40-50 bp, and the annealing temperature of the bases in the overlapping region was above 60°C.

[0111] (2) The first round of PCR amplification reaction system: KAPA Taq ReadyMix (KK1006, Merck) 10 μL, upstream and downstream primers 0.5 μL each, ddH2O 7 μL, template 1 μL, total reaction system 20 μL; the primer sequences are shown in Table 2.

[0112] The reaction conditions for the first round of PCR amplification were: 98°C for 1 min; 98°C for 30 s, 55-60°C for 30 s, 72°C for 1-4 min, 15 cycles; 72°C for 10 min.

[0113] (3) Take 1 μL of the first-round PCR amplification reaction solution as the template for the second-round PCR amplification reaction. The second-round PCR amplification reaction system: 10 μL of KAPA Taq ReadyMix (KK1006, Merck), 0.5 μL of upstream and downstream primers, 7 μL of ddH2O, 1 μL of template, and the total reaction system is 20 μL; the primer sequences are shown in Table 2.

[0114] The reaction conditions for the second round of PCR amplification were as follows: 98°C for 1 min; 98°C for 30 s, 50-60°C for 30 s, 72°C for 1-4 min, 32 cycles; 72°C for 10 min.

[0115] (4) The target fragment was recovered by gel electrophoresis and connected to the pEASY-Blunt subcloning vector (CB101, Quanshijin Biotechnology Co., Ltd.) for sequencing.

[0116] According to the above steps, the following six modules are constructed:

[0117] (a) GAL1p, tHMGR and ADH1t were connected to construct the gene expression module GAL1p-tHMGR-ADH1t, which was named module I. The primers for cloning GAL1p in the first round of PCR were I-F1 and I-R1, the primers for cloning tHMGR were I-F2 and I-R2, and the primers for cloning ADH1t were I-F3 and I-R3; the primers for the second round of PCR were I-F1 and I-R3; the primer sequences are shown in Table 2.

[0118] (b) GAL10p, UPC2-1 and CYC1t were connected to construct the gene expression module GAL10p-UPC2-1-CYC1t, named module II. The primers for cloning GAL10p in the first round of PCR were II-F1 and II-R1, the primers for cloning UPC2-1 were II-F2 and II-R2, and the primers for cloning CYC1t were II-F3 and II-R3; the primers for the second round of PCR were II-F1 and II-R3; the primer sequences are shown in Table 2.

[0119] (c) Connecting module I and module II, constructing the gene expression module GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t, named module III. The primers for cloning module I in the first round of PCR were III-I-F1 and III-I-R1, and the primers for cloning module II were III-II-F2 and III-II-R2; the primers for the second round of PCR were III-I-F1 and III-II-R2; the primer sequences are shown in Table 2.

[0120] (d) GAL10p, Erg20 F96W-N127W Connected with CYC1t to construct the gene expression module GAL10p-Erg20 F96W -N127W -CYC1t, named module IV. Among them, the primers for cloning GAL10p in the first round of PCR were IV-F1 and IV-R1, and the primers for cloning Erg20 F96W-N127W The primers for cloning CYC1t were IV-F2 and IV-R2, and the primers for cloning CYC1t were IV-F3 and IV-R3; the primers for the second round of PCR were IV-F1 and IV-R3; the primer sequences are shown in Table 2.

[0121] (e) The HIS screening marker and module IV were connected to construct a gene expression module HIS-V, which was named module V; wherein the primers for cloning the screening marker HIS in the first round of PCR were V-F1 and V-R1, and the primers for cloning module IV were V-F2 and V-R2; the primers for the second round of PCR were V-F1 and V-R2; the primer sequences were shown in Table 2.

[0122] (f) GAL1p, CoTPS1 and ADH1t were connected to construct the gene expression module GAL1p-CoTPS1-ADH1t, named module VI. The primers for cloning GAL1p in the first round of PCR were VI-F1 and VI-R1, the primers for cloning CoTPS1 were VI-F2 and VI-R2, and the primers for cloning ADH1t were VI-F3 and VI-R3; the primers for the second round of PCR were VI-F1 and VI-R3; the primer sequences are shown in Table 2.

[0123] The UPC2-1 gene is a PCR-based site-directed mutagenesis technique that mutates the transcription factor gene UPC2 into UPC2-1, and its nucleotide sequence is shown in SEQ ID NO.3; the nucleotide sequences of the tHMGR and UPC2 genes are shown in SEQ ID NO.3-4, respectively; the Erg20 F96W-N127W It is obtained by site-directed mutation of Erg20, and its nucleotide sequence is shown in SEQ ID NO.5; the nucleotide sequence of the Erg20 gene is shown in SEQ ID NO.6; the nucleotide sequences of GAL1p, GAL10p, ADH1t and CYC1t are shown in SEQ ID NO.7-10 respectively; the nucleotide sequence of the screening marker HIS is shown in SEQ ID NO.11 respectively.

[0124] S2. Construction of yeast genome-integrating expression vector pCfB2875-III

[0125] (1) The vector pCfB2875 (Addgene) was digested with restriction endonuclease SfaAI. The digestion system was as follows: 1 μL of pCfB2875 vector, 0.5 μL of restriction endonuclease SfaAI (FD2094, Merck), 1 μL of 10x Fast Digest Buffer, 7.5 μL of ddH2O, and the total reaction system was 10 μL.

[0126] (2) Enzyme digestion was performed at 37°C for 1 h, and then the linearized vector was recovered by gel extraction.

[0127] (3) Module III was amplified from pEASY-Blunt-GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t (i.e., the above module III was connected to the pEASY-Blunt vector), the purified PCR product was recovered by gel recovery, and then homologously recombined into the linearized pCfB2875 vector between the restriction endonuclease cutting sites SfaAI and NheI. The homologous recombination system is as follows: II 1μL, 5×CE II Buffer 2μL, recovered linearized vector 1μL, purified PCR product 4μL, ddH2O 2μL, the total reaction system is 10μL.

[0128] (4) The cells were reacted at 37°C for 30 min, and the homologous recombination system was transformed into DH5α competent cells. The cells were spread on plates and cultured at 37°C overnight. The integrative expression vector pCfB2875-III was obtained by colony PCR identification and plasmid sequencing.

[0129] S3. Construction of yeast genome-integrating expression vector pCfB2797-VI

[0130] (1) The vector pCfB2797 (Addgene) was digested with restriction endonuclease SfaAI. The digestion system was as follows: 1 μL of pCfB2797 vector, 0.5 μL of restriction endonuclease SfaAI (FD2094, Merck), 1 μL of 10x Fast Digest Buffer, 7.5 μL of ddH2O, and the total reaction system was 10 μL.

[0131] (2) Enzyme digestion was performed at 37°C for 1 h, and then the linearized vector was recovered by gel extraction.

[0132] (3) Module VI was amplified from pEASY-Blunt-GAL1p-CoTPS1-ADH1t (i.e., the module VI was connected to the pEASY-Blunt vector), and the purified PCR product was recovered by gel recovery, and then homologously recombined into the restriction endonuclease cutting site SfaAI of the linearized pCfB2797 vector. The homologous recombination system is as follows: II 1μL, 5×CEII Buffer 2μL, recovered linearized vector 1μL, purified PCR product 4μL, ddH2O 2μL, the total reaction system is 10μL.

[0133] (4) The cells were reacted at 37°C for 30 min, and the homologous recombination system was transformed into DH5α competent cells. The cells were spread on plates and cultured at 37°C overnight. The integrative expression vector pCfB2797-VI was obtained by colony PCR identification and plasmid sequencing.

[0134] (5) The vector pCfB2797-VI was digested with restriction endonuclease NheI. The digestion system was as follows: 1 μL of pCfB2797-VI vector, 0.5 μL of restriction endonuclease NheI (FD2094, Merck), 1 μL of 10x Fast Digest Buffer, 7.5 μL of ddH2O, and the total reaction system was 10 μL.

[0135] (6) Enzyme digestion was performed at 37°C for 1 h, and then the linearized vector was recovered by gel extraction.

[0136] (7) Module VI was amplified from pEASY-Blunt-GAL1p-CoTPS1-ADH1t (i.e., the module VI was connected to the pEASY-Blunt vector), and the purified PCR product was recovered by gel recovery, and then homologously recombined into the restriction endonuclease cutting site SfaAI of the linearized pCfB2797-VI vector. The homologous recombination system is as follows: II 1μL, 5×CE II Buffer 2μL, recovered linearized vector 1μL, purified PCR product 4μL, ddH2O 2μL, the total reaction system is 10μL.

[0137] (8) The cells were reacted at 37°C for 30 min, and the homologous recombination system was transformed into DH5α competent cells. The cells were spread on plates and cultured at 37°C overnight. The integrative expression vector pCfB2797-VI was obtained by colony PCR identification and plasmid sequencing.

[0138] Table 2 Primer sequences used to construct each module in the present invention

[0139]

[0140]

[0141] Example 4: Construction of engineered yeast

[0142] like Figure 1 As shown, this embodiment provides a method for constructing an engineered strain of Saccharomyces cerevisiae for synthesizing geraniol, which specifically comprises the following steps:

[0143] S1. Construction of Saccharomyces cerevisiae strain XY01

[0144] (1) The constructed integrative expression vector pCfB2875-III was digested with restriction endonuclease NotI, and the target band was detected and recovered by gel electrophoresis to obtain the DNA integration fragment S1.

[0145] (2) Take 50 μL of the purified DNA fragment and transform the Saccharomyces cerevisiae engineering strain BY4741 to integrate the module into the TY3 site of the yeast BY4741 chromosome, and screen it using a uracil-deficient medium (SD-URA medium) plate. After colony PCR verification, the Saccharomyces cerevisiae engineering strain XY01 was obtained.

[0146] S2. Construction of Saccharomyces cerevisiae engineered strain XY02

[0147] (1) The pSH65 vector (Wuhan Miaoling Biotechnology Co., Ltd.) was transformed into the Saccharomyces cerevisiae engineered strain XY02 and screened using YPD solid medium containing 100 μg / mL bleomycin.

[0148] (2) Single clones were picked and cultured in YPD medium containing 100 μg / mL bleomycin.

[0149] (3) After four days of culture, monoclonal colonies were obtained by streak dilution method.

[0150] (4) The genome of the monoclonal colony was extracted, and the integrated DNA fragment S1 was amplified by PCR. The integrated vector KIURA3 screening marker was knocked out by sequencing screening, and then the strain was subcultured in YPD medium for ten days, and the pSH65 plasmid was discarded to obtain the Saccharomyces cerevisiae engineered strain XY02.

[0151] S3. Construction of Saccharomyces cerevisiae engineered strain XY03

[0152] (1) Module V was removed from pEASY-Blunt-HIS-GAL10p-Erg20 F96W-N127W -CYC1t was amplified, and the PCR reaction system was: KAPA Taq ReadyMix (KK1006, Merck) 25 μL, upstream and downstream primers 1.25 μL each, ddH2O 21.5 μL, template 1 μL, and the total reaction system was 50 μL.

[0153] The PCR amplification reaction conditions were as follows: 98°C for 1 min; 98°C for 30 s, 55-60°C for 30 s, 72°C for 2 min, 32 cycles; 72°C for 10 min.

[0154] The PCR products were detected by electrophoresis and purified and recovered.

[0155] (2) The recovered PCR fragment was transformed into the Saccharomyces cerevisiae engineering strain XY02, and screened using a methionine (SD-HIS medium) plate to obtain the Saccharomyces cerevisiae engineering strain XY03.

[0156] S4. Construction of the engineered yeast strain XY04 for synthesizing geraniol

[0157] (1) The constructed integrative expression vector pCfB2797-VI was digested with restriction endonuclease NotI, and the target band was detected and recovered by gel electrophoresis to obtain the DNA integration fragment S2.

[0158] (2) 50 μL of the purified DNA fragment was taken and transformed into the Saccharomyces cerevisiae engineering strain XY03 to integrate the module into the TY2 site of the chromosome of the Saccharomyces cerevisiae engineering strain XY03, and screened using a uracil-deficient medium (SD-HIS-URA medium) plate. After colony PCR verification, a Saccharomyces cerevisiae engineering strain XY04 with high santalol production was obtained.

[0159] Example 4

[0160] This embodiment provides a method for producing santalol by fermentation using an engineered strain of Saccharomyces cerevisiae XY04, which specifically comprises the following steps:

[0161] S1. Fermentation culture of Saccharomyces cerevisiae engineered strain XY04

[0162] (1) A single colony of the engineered Saccharomyces cerevisiae strain XY04 was inoculated into a test tube containing 5 mL of YPD and cultured at 220 rpm at 30°C until the OD value reached 2-3.

[0163] (2) Take 1 mL of the above bacterial solution and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of YPD medium. Cultivate at 220 rpm at 30°C for 168 h to obtain a fermentation broth.

[0164] S2. Extraction and detection of target product

[0165] (1) Take the above fermentation broth, add an equal volume of ethyl acetate, sonicate for 1 h, and then let stand for 48 h.

[0166] (2) Take the organic layer into a clean liquid phase vial and perform GC-MS detection. The instrument used is preferably Agilent GC-MS 7890B-5977B. The detection method is as follows: the injection volume is 1 μL, the solvent delay is set to 12 min, the carrier gas is helium, and the flow rate is 1 mL / min. The chromatographic column is HP-5MS. Chromatographic conditions: 50°C, 3 min; heating at a rate of 20°C / min to 70°C, 1 min; heating at 15°C / min to 300°C, 3 min.

[0167] (3) GC-MS test results showed that the engineered strain of Saccharomyces cerevisiae XY04 could produce 75.26 mg / L of geraniol after 168 h of fermentation.

[0168] The present invention successfully cloned and identified a new geraniol synthase gene CoTPS1 through a synthetic biology strategy, and its sequence is shown in SEQ ID NO.1. To verify the catalytic function of the gene, the present invention cloned the CoTPS1 gene into a yeast expression vector and introduced it into an engineered strain of Saccharomyces cerevisiae through transformation technology. Under optimized culture conditions, the recombinant strain was fermented, and gas chromatography-mass spectrometry (GC-MS) technology was used to analyze metabolites (such as Figure 1 and Figure 2 The test results showed that the engineered strain was able to synthesize geraniol, further proving that CoTPS1 had the catalytic activity of geraniol synthase, laying the foundation for the construction of microbial cell factories.

[0169] On this basis, the present invention further optimizes the metabolic pathway of Saccharomyces cerevisiae to improve the biosynthetic capacity of geraniol. First, through the regulation of the mevalonic acid (MVA) pathway, tHMGR (a mutant encoding the rate-limiting enzyme HMG-CoA reductase) and UPC2-1 (a transcription factor mutant that can enhance the synthesis of isoprenoid compounds) are overexpressed to improve the supply capacity of precursor substances. Secondly, in the monoterpene biosynthesis pathway, a specific mutant farnesyl pyrophosphate synthase (Erg20F96W-N127W) is introduced to optimize the metabolic flow of geraniol pyrophosphate (GPP), making it more conducive to the synthesis of geraniol. Finally, in this optimized metabolic background, CoTPS1 is heterologously expressed to ensure the efficient synthesis of geraniol. The schematic diagram of the construction of the engineered strain is shown in the following figure. Figure 3 shown.

[0170] In order to further evaluate the geraniol production capacity of the optimized Saccharomyces cerevisiae engineered strain, the present invention adopts a batch fermentation process to systematically culture the recombinant strain and monitor the geraniol production. The fermentation process adopts an optimized carbon source supply strategy, using glucose or glycerol as the main carbon source, and controlling oxygen supply and pH to improve the biosynthesis efficiency. After a certain period of fermentation, the culture solution is extracted with an organic solvent, and the product concentration is analyzed using GC-MS. The results show that the final geraniol production reaches 75.26 mg / L (such as Figure 4 As shown in the figure, the biosynthesis capacity of geraniol was significantly improved compared with that of the non-engineered strain, which verifies the effectiveness of the optimization strategy proposed in the present invention in improving the biosynthesis capacity of geraniol.

[0171] The technical solution of the present invention breaks through the bottleneck of plant extraction and traditional microbial metabolic engineering in the synthesis of geraniol, and realizes the full-process design of gene cloning, metabolic pathway optimization and fermentation process optimization. Compared with the chemical synthesis method, the present invention provides a green and sustainable microbial manufacturing path, which significantly reduces the production cost and improves the purity and yield of geraniol. In addition, this technology platform can not only be used for the synthesis of geraniol, but also can be extended to the biosynthesis of other high value-added terpenoids (such as nerol, linalool, etc.), which has important industrial application value. In the future, by further optimizing metabolic flow regulation, enzyme engineering transformation and biological process amplification, it is expected to achieve industrial-scale production of geraniol, providing a better source of raw materials for food, flavors and fragrances, medicine and other fields.

[0172] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A geraniol synthase gene CoTPS1, characterized in that The nucleotide sequence is shown in the sequence list SEQ ID NO.1; the amino acid residue sequence of the gene-encoded protein is shown in the sequence list SEQ ID NO.

2.

2. A method for cloning the geraniol synthase gene CoTPS1 as claimed in claim 1, characterized in that: The specific steps are as follows: (1) Extracting total RNA from Celastrus orcidudata and reversely transcribing it into cDNA; (2) Using cDNA as a template, specific primers were designed and PCR was performed to amplify the geraniol synthase gene CoTPS1; (3) Recovering and purifying the PCR product in step (2) by agarose gel electrophoresis and an agarose gel DNA recovery kit to obtain the geraniol synthase gene CoTPS1 fragment.

3. The method for cloning the geraniol synthase gene CoTPS1 according to claim 2, characterized in that: PCR amplification was performed using 2xPhanta Max Master Mix high-fidelity DNA polymerase.

4. The method for cloning the geraniol synthase gene CoTPS1 according to claim 2, characterized in that: The PCR amplification system was 2x Phanta Max Master Mix 10 μL, forward and reverse primers 0.5 μL each, ddH2O 8 μl, template 1 μL, and the total reaction system was 20 μL; The PCR amplification reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 1 min, 32 cycles; 72°C for 5 min.

5. A method for using the geraniol synthase gene CoTPS1 as claimed in claim 1, characterized in that: An engineered bacterium for synthesizing geraniol is constructed, wherein the strain is based on Saccharomyces cerevisiae as a starting strain, overexpresses tHMGR, UPC2-1, Erg20 F96W -N127W and CoTPS1; wherein: (1) tHMGR and UPC2-1 genes were integrated into the TY3 locus of the Saccharomyces cerevisiae chromosome; (2) Erg20 F96W-N127W Integrated into the ROX1 locus; (3) The CoTPS1 gene was integrated into the TY2 locus of the Saccharomyces cerevisiae chromosome.

6. The application method of the geraniol synthase gene CoTPS1 as claimed in claim 5, wherein the cerevisiae yeast is cerevisiae yeast BY4741.

7. The application method of the geraniol synthase gene CoTPS1 as claimed in claim 5, and the construction method of the geraniol synthesizing yeast engineering strain comprises the following steps: (1) Use overlapping PCR to construct the following modules: (a) GAL1p, tHMGR and ADH1t were connected to construct the gene expression module GAL1p-tHMGR-ADH1t, named module I; (b) GAL10p, UPC2-1 and CYC1t were connected to construct the gene expression module GAL10p-UPC2-1-CYC1t, which was named module II; (c) connecting module II and module III to construct the gene expression module GAL1p-tHMGR-ADH1t-GAL10p-UPC2-1-CYC1t, named module III; (d) GAL10p, Erg20 F96W-N127W Connected with CYC1t to construct the gene expression module GAL10p-Erg20 F96W-N127W -CYC1t, named module IV; (e) Template IV and HIS selection marker are connected to construct the gene expression module HIS-V, which is named module V; (f) GAL1p, CoTPS1 and ADH1t were connected to construct the gene expression module GAL1p-CoTPS1-ADH1t, named module VI; (2) Construction of strain XY01 Insert module III into the SfaSI restriction site of vector pCfB2875 to obtain the integration expression vector pCfB2875-III; then cut the integration expression vector pCfB2875-III with restriction endonuclease NotΙ to obtain DNA integration fragment S1; then integrate DNA integration fragment S1 into the TY3 site of the chromosome of yeast engineering bacteria to obtain strain XY01; (3) Construction of strain XY02 The pSH65 vector was transformed into strain XY01, and then screened to obtain strain XY02; (4) Construction of strain XY03 Transform the module V into the yeast engineering strain XY02 to obtain the strain XY03; (5) Construction of strain XY04 The module VI is inserted into the SfaSI restriction site of the vector pCfB2797 to obtain the integrated expression vector pCfB2797-VI; the module IX is inserted into the NheI restriction site of the vector pCfB2797-VI to obtain the integrated expression vector pCfB2797-VI-IX; the integrated expression vector pCfB2797-VI-IX is then cut with the restriction endonuclease NotΙ to obtain the DNA integrated fragment S2; the DNA integrated fragment S2 is then integrated into the TY2 site of the chromosome of the strain TX04 to obtain the strain XY04, and the Saccharomyces cerevisiae engineered strain with high santalol production is obtained.

8. The method for using the geraniol synthase gene CoTPS1 as claimed in claim 5, wherein the tHMGR, UPC2-1 and Erg20 F96W-N127W The gene can be cloned from the genome of Saccharomyces cerevisiae BY4741, and its nucleotide sequence is shown in SEQ ID NO.3-5; The GAL1p and GAL10p described in step (1) are promoter sequences, which can be cloned from the genome of Saccharomyces cerevisiae BY4741, and their nucleotide sequences are shown in SEQ ID NOs. 7 to 8 respectively; The ADH1t and CYC1t described in step (1) are terminator sequences, which can be cloned from the genome of Saccharomyces cerevisiae BY4741, and their nucleotide sequences are shown in SEQ ID NOs. 9 to 10 respectively; The nucleotide sequence of the screening marker HIS described in step (1) is shown in SEQ ID NO.

11.

9. The application method of the geraniol synthase gene CoTPS1 as claimed in claim 5, wherein the geraniol synthesizing yeast engineering bacteria is activated and inoculated into a fermentation medium for fermentation culture to obtain geraniol.

10. Use of the geraniol synthase gene CoTPS1 as claimed in claim 1 in the construction of an engineered strain of Saccharomyces cerevisiae that produces geraniol and other terpenoid compounds with geraniol as a substrate.

Images