A method and application of heterologous synthesis of taxadiene using Chlamydomonas reinhardtii chloroplasts

Abstract

The present invention discloses a method and application of heterologous synthesis of taxadiene by using Chlamydomonas reinhardtii chloroplasts, and belongs to the field of bioengineering technology. The present invention provides a taxadiene synthase gene (TS) that can be heterologously expressed in Chlamydomonas reinhardtii chloroplasts, and constructs a recombinant expression vector or a recombinant algae strain containing the TS gene for synthesizing taxadiene. The present invention also uses the recombinant algae strain as a production algae strain, uses a biphasic culture method, and controls the culture conditions to promote the biosynthesis of taxadiene by doubling acetate, adding methyl jasmonate, and controlling the culture conditions, so that the taxadiene yield per unit volume can reach 185.5±4.4μg / L, and the yield per unit cell can reach 5.6±0.2fg / cell.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method and application of heterologous synthesis of taxadiene by utilizing Chlamydomonas reinhardtii chloroplasts. Background Art

[0002] Isoprenoids, also known as terpenes or terpenoid compounds, are the largest family of natural products. There are a large number of products with high commercial value from isoprenoids, such as pigments, perfumes and cosmetics, drugs, as well as chemical signals and pest control agents. At the same time, many secondary metabolites derived from plants have important pharmaceutical significance, such as artemisinin and paclitaxel. Among them, paclitaxel is widely used as an anticancer drug to treat ovarian cancer, breast cancer and lung cancer, as well as lymphoma and leukemia. In addition to pharmacological effects, some terpene products have completed mature commercial development, including health products, cosmetics, food and beverages, biofuels and rubber-derived biomaterials. Corresponding to the wide range of uses of isoprenoids, the production of terpenes has severely limited the progress of their commercial development. Terpene products with important biological activity are often very low in content or difficult to extract in natural raw materials, and are not economically feasible. Although chemical synthesis avoids damage to plants and the natural environment, the complex structure of isoprenoids means that most terpenes and isoprenoids cannot be chemically synthesized. At the same time, high production costs and industrial toxic byproducts still hinder the large-scale production of terpenoids. The production of isoprenoids urgently needs to develop an economical, efficient and sustainable approach.

[0003] As an emerging green cell factory, Chlamydomonas reinhardtii has the characteristics of fast growth, clear genetic background, and strong and flexible supply capacity of terpene precursors. At the same time, chloroplast engineering has many advantages over nuclear genetic engineering, such as stable and efficient expression, avoiding gene silencing, and no need for continuous use of antibiotics for screening. Although in theory, chloroplast engineering can better utilize the terpene production potential of Chlamydomonas reinhardtii, however, no relevant successful cases have been seen in Chlamydomonas reinhardtii. In previous studies, cis-abienol (diterpene) synthase was successfully overexpressed in the chloroplasts of Chlamydomonas reinhardtii, but the synthesis of the target product was not detected. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide a taxadiene synthase gene heterologously expressed in the chloroplasts of Chlamydomonas reinhardtii, which can be used to construct a corresponding recombinant expression vector or a recombinant algae strain for synthesizing taxadiene.

[0005] The present invention also aims to provide a method for producing taxadiene, thereby increasing the biosynthesis of taxadiene in the recombinant algae strain.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a taxadiene synthase gene heterologously expressed in chloroplasts of Chlamydomonas reinhardtii, wherein the nucleotide sequence of the gene is SEQ ID NO:1.

[0008] The present invention provides application of the above gene in construction of recombinant algae strain of Chlamydomonas reinhardtii.

[0009] The present invention provides a recombinant expression vector containing the gene, wherein the recombinant expression vector comprises a pASapI recombinant expression vector and a pSRSapI recombinant expression vector; the pASapI recombinant expression vector contains an atpA promoter, and the pSRSapI recombinant expression vector contains a psaA promoter.

[0010] Preferably, a codon ATG is added to the 5' end of the taxadiene synthase gene in the pSRSapI recombinant expression vector.

[0011] The present invention provides a recombinant algae strain of Chlamydomonas reinhardtii containing the above gene or the above recombinant expression vector.

[0012] Preferably, the recombinant algae strains include the recombinant algae strain TSA with a preservation number of CCTCC M 20221695, the recombinant algae strain TSPM with a preservation number of CCTCC M 20221696, and the recombinant algae strain TSP with a preservation number of CCTCC M20221697.

[0013] The present invention provides a method for producing taxadiene. The above-mentioned recombinant algae strain is used as a production algae strain, dodecane is added to a TAP medium for dual-phase culture, and taxadiene is obtained.

[0014] Preferably, the culture conditions are continuous illumination, the culture temperature is 15-30°C, and the inoculation density of the recombinant algae strain is 1×10 6 ~10×10 6 cells / mL.

[0015] Preferably, acetate and / or methyl jasmonate are added to the TAP medium.

[0016] The present invention provides the application of the above recombinant algae strain or the above method for producing taxadiene in the chemical or pharmaceutical fields.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention obtains a taxadiene synthase gene that can be heterologously expressed in Chlamydomonas reinhardtii chloroplasts through gene optimization, and selects atpA promoter, psaA promoter or psaA promoter+gene nitrogen end optimization to construct corresponding expression vectors and engineered algae strains, thereby achieving the expression and accumulation of taxadiene synthase protein.

[0019] The present invention provides a method for efficiently producing taxadiene using the engineered algae strain of the present invention by adjusting the acetate concentration of the culture medium, the illumination conditions, the culture temperature, the inoculation density and the selection of exogenous substances. The yield per unit volume of taxadiene can reach 185.5±4.4 μg / L, and the yield per unit cell can reach 5.6±0.2 fg / cell.

[0020] Recombinant algae strain TSA (Latin name Chlamydomonas reinhardtiiTAS), depository: China Center for Type Culture Collection (CCTCC for short), depository address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20221695, deposit date is October 31, 2022.

[0021] The recombinant algal strain TSPM (Latin name Chlamydomonas reinhardtii TSPM), depository: China Center for Type Culture Collection (CCTCC for short), depository address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20221696, deposit date is October 31, 2022.

[0022] Recombinant algae strain TSP (Latin name Chlamydomonas reinhardtii TSP), depository: China Center for Type Culture Collection (CCTCC), depository address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M20221697, deposit date is October 31, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Overexpression vector map, a is the pASapI overexpression vector map, b is the pSRSapI overexpression vector map;

[0024] Figure 2 : Detection results of taxadiene synthase protein products in transformed algae strains;

[0025] Figure 3 : Detection results of the accumulation of taxadiene synthase protein products in transformed algae strains;

[0026] Figure 4: Identification spectrum of dual-phase culture products, a is the taxadiene yield of TSA algae, b is the taxadiene yield of TSP algae, c is the taxadiene yield of TSPM algae, d is the characteristic ion peak of taxadiene;

[0027] Figure 5 :Effects of culture conditions on taxadiene production, a is the effect of different acetate concentrations and light conditions on taxadiene production, b is the effect of different acetate concentrations and culture temperatures on taxadiene production; c is the effect of different acetate concentrations and inoculation density / exogenous additives on taxadiene production;

[0028] Figure 6 : Yield curve of PM1 transformants under optimal culture conditions. DETAILED DESCRIPTION

[0029] The invention provides a taxadiene synthase gene (TS) heterologously expressed in a chloroplast of Chlamydomonas reinhardtii. The nucleotide sequence of the gene is SEQ ID NO: 1, which is obtained by optimizing the codon usage preference of the chloroplast of Chlamydomonas reinhardtii and mRNA secondary structure and adding a 3×HA tag to a taxadiene synthase gene (downloaded from Genebank: Accession no. U48796), and the nucleic acid sequence is 2676 bp long; the corresponding amino acid sequence is SEQ ID NO: 2, and the obtained taxadiene synthase can synthesize taxadiene by using GGPP.

[0030] The present invention also provides the application of the above gene in the construction of recombinant algae strains of Chlamydomonas reinhardtii.

[0031] The present invention provides a recombinant expression vector containing the gene, wherein the recombinant expression vector comprises a pASapI recombinant expression vector and a pSRSapI recombinant expression vector.

[0032] The pASapI recombinant expression vector of the present invention contains atpA promoter, and the TS fragment is inserted between atpA promoter and 3×HA of the pASapI vector by double digestion with restriction endonucleases SapI and PstI to obtain a pASapI recombinant expression vector, which is recorded as pASapI-TS recombinant expression vector. The pASapI plasmid vector of the present invention is composed of the chloroplast atpA5'UTR of Chlamydomonas reinhardtii, rbcL 3'UTR, Amp resistance gene and psbH upstream and downstream recombination element regions, wherein the recombination region contains the complete sequence of the psbH gene. The map of pASapI overexpression vector is shown in Figure 1 -a.

[0033] The pSRSapI recombinant expression vector of the present invention contains a psaA promoter. The psaA-exon1 sequence (GenBank: Accession No. nm006516) and the TS fragment are treated with SapI enzyme digestion and connected to obtain a fragment psaA-TS. Then, the psaA-TS fragment replaces the region between MluI and BamHI of the plasmid pASapI to obtain the pSRSapI recombinant expression vector, which is recorded as a pSRSapI-TS recombinant expression vector.

[0034] The pSRSapI recombinant expression vector of the present invention optimizes the 5' end of the TS gene, which can effectively improve the anti-degradation ability of the synthesized TS protein. The optimization method is: add a methionine codon ATG to the 5' end of the TS gene, and use the pSRSapI-TS recombinant expression vector for PCR amplification to construct the pSRSapI-TSM recombinant expression vector. The map of the pSRSapI overexpression vector is shown in Figure 1 -b.

[0035] The recombinant expression vector of the present invention can complete the psbH gene complementation through homologous recombination, restore the photosynthetic autotrophic ability of the Chlamydomonas reinhardtii strain, and integrate the target gene into the chloroplast genome to achieve the expression and accumulation of taxadiene synthase protein.

[0036] The present invention also provides a recombinant algae strain of Chlamydomonas reinhardtii containing the above-mentioned gene or recombinant expression vector. Preferably, the recombinant algae strain of the present invention includes the recombinant algae strain TSA with a preservation number of CCTCC M 20221695, the recombinant algae strain TSPM with a preservation number of CCTCC M20221696, and the recombinant algae strain TSP with a preservation number of CCTCC M20221697. The recombinant algae strain TSA contains the pASapI-TS recombinant expression vector, the recombinant algae strain TSP contains the pSRSapI-TS recombinant expression vector, and the recombinant algae strain TSPM contains the pSRSapI-TSM recombinant expression vector. The recombinant algae strain of the present invention is preferably constructed with Chlamydomonas reinhardtii CC5168 as the host.

[0037] The method for constructing the recombinant Chlamydomonas reinhardtii strain of the present invention comprises: linearizing the recombinant expression vector by Eco147I enzyme digestion, transforming the linearized plasmid into Chlamydomonas reinhardtii by glass bead method, and then screening out the engineered algae strain capable of restoring photoautotrophic growth by high-salt culture medium.

[0038] The present invention also provides a method for producing taxadiene, wherein the recombinant algae strain of the present invention is used as a production algae strain, dodecane is added to a TAP medium for dual-phase culture, and taxadiene is obtained.

[0039] The content of dodecane in the dual-phase culture system of the present invention is preferably 8-12%, more preferably 10%. The culture condition of the present invention is continuous illumination, and the illumination intensity is preferably 70 μmol / (m 2 ·s); the culture temperature is 15-30°C, preferably 15-20°C; the inoculation density of the recombinant algae strain is 1×10 6 ~10×10 6 cells / mL, preferably 7×10 6 cells / mL.

[0040] The TAP medium of the present invention adds high concentration of acetate, and based on the acetate concentration in the TAP medium, the TAP medium is additionally added with 1 to 6 times of acetate, preferably 2 to 5 times of acetate; the increase in the acetate concentration is achieved by increasing the amount of Tris-Acetate added to the TAP medium. The present invention can significantly upregulate the expression level of ABC transporters by adding acetate, so that the CrABCG3 and CrABCG4 genes are continuously upregulated until the end of the culture, thereby promoting the synthesis of taxadiene.

[0041] The TAP medium of the present invention adds 0.4-0.6 mM methyl jasmonate, and the addition amount of methyl jasmonate is preferably 0.5 mM. The synthesis of taxadiene can be further promoted by adding methyl jasmonate.

[0042] The present invention also provides the application of the above recombinant algae strain or the above method for producing taxadiene in the chemical or pharmaceutical fields.

[0043] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] In a specific embodiment of the present invention, Chlamydomonas reinhardtii CC5168 (TN72) and CC124, and plasmid pASapI were purchased from the Chlamydomonas Genetics Center of Duke University, USA; the TS nucleotide sequence (SEQ ID NO: 1) was delivered to Universal Biotechnology (Anhui, China) for synthesis.

[0045] Tris-acetate phosphate (TAP) medium: Tris-Acetate 10mL, Phosphate buffer 10mL, Beijerinck's Solution 10mL, Hutner's trace elements Solution 1mL, dilute to 1L. Tris-Acetate: Tris 242g, glacial acetic acid 100mL, dilute to 1L; Phosphate buffer: K 2 HPO 4 11.92g, KH 2 PO 4 6.05 g, dilute to 1 L; Beijerinck's solution: NH 4 Cl 40g, MgSO 4 7H 2 O 10g, CaCl 2 ·2H 2 O 5g, make up to 1L.

[0046] High salt basal (HSM) medium: HSM Phosphate buffer 5.0mL, HSM Beijerinck's Solution 5.0mL, Hutner's trace elements Solution 1.0mL, dilute to 1L. HSM Phosphate buffer: NH 4 Cl 100g, MgSO 4 7H 2 O 4g, CaCl 2 ·2H 2 O 2g, dilute to 1L. HSM Beijerinck's Solution: K 2 HPO 4 288g, KH 2 PO 4 144 g, make up to 1 L.

[0047] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1

[0050] 1. Construction of pASapI-TS recombinant expression vector

[0051] The TS gene (SEQ ID NO: 1) and the pASapI vector were treated with double restriction enzymes SapI and PstI. The restriction enzyme system was as follows: 1 μL SapI endonuclease, 1 μL PstI endonuclease, 2 μL 10× fast enzyme buffer, 1 μg TS fragment / pASapI vector, ddH 2 O up to 20μL; reaction conditions: incubate at 37℃ for 1h, inactivate at 80℃ for 5min. The digestion products were identified by gel electrophoresis, and the larger fragments of the pASapI digestion products, TS digestion products, were recovered by gel excision. The recovered TS and pASapI fragments were connected by DNA ligase, and the connection system was: TS fragment 1.7μL (0.2μg), pASapI fragment 6.8μL (0.6μg), 10×T4 DNA ligase buffer 1μL, T4 DNA ligase 0.5μL. The reaction was carried out in a constant temperature water bath at 16℃ for 4h to obtain the target plasmid pASapI-TS.

[0052] 2. Construction of pSRSapI-TS recombinant expression vector

[0053] (1) Cloning of psaA-exon1 fragment

[0054] Using Chlamydomonas reinhardtii CC124 total DNA as template, primers psaA-F (SEQ ID NO: 3): 5'-GTTCACGCGTAAGCTTTCTTAATTCAACATTT-3' and psaA-R (SEQ ID NO: 4): 5'-ATAGGCTCTTCTCATGGATTTCTCCTTATAATAAC-3' were used to amplify the psaA-exon1 fragment. The reaction system included KOD OneTM PCR Master Mix-Blue-25 μL, psaA-F (10 μM) 1.5 μL, psaA-R (10 μM) 1.5 μL, CC124 genome 200 ng, ddH 2 O up to 50μL. PCR amplification conditions are as follows: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 47℃ annealing for 5s, 68℃ extension for 15s, 35 cycles, and 68℃ extension for 3min. After the program is completed, the detection is carried out by gel electrophoresis, and the 290bp target band is recovered by cutting the gel.

[0055] (2) Cloning of psaA-TS fragment

[0056] The obtained psaA-exon1 fragment and TS gene (SEQ ID NO: 1) fragment were treated with SapI restriction enzyme digestion. The single enzyme digestion system was: SapI 2 μL, 10× fast digestion enzyme buffer 2 μL, psaA-exon1 fragment / TS fragment 2 μg, ddH 2O Up to 20μL. Reaction conditions: incubate at 37℃ for 1h. The reaction product was directly recovered using a gel recovery kit. The psaA-exon1 and TS fragments were integrated using T4 ligase. The ligation system was: 1.5μL of psaA-exon1 fragment, 7μL of TS fragment, 1μL of 10×T4 DNA ligase buffer, and 0.5μL of T4 DNA ligase. The reaction system was placed in a 16℃ constant temperature water bath for 4h to obtain a psaA-TS fragment with a size of 3.0kb.

[0057] (3) Construction of pSRSapI-TS

[0058] The psaA-TS fragment was used to replace the region between MluI and BamHI of the plasmid pASapI through double enzyme digestion. The enzyme digestion system was as follows: MluI 2μL, BamHI 2μL, 10× fast digestion enzyme buffer 1μL, pASapI / psaA-TS 2μg, ddH 2 O Up to 20μL. Incubate the reaction system at 37℃ for 1h. Identify the results of enzyme cleavage by electrophoresis, cut out the larger band in the pASapI enzyme cleavage reaction product, and directly recover it with the psaA-TS enzyme cleavage system. Use T4 ligase to integrate the enzyme fragments. Ligation system: psaA-TS fragment 1.7μL (0.2μg), pASapI fragment 6.8μL (0.6μg), 10×T4 DNA ligase buffer 1μL, T4 DNA ligase 0.5μL. React in a constant temperature water bath at 16℃ for 4h to obtain the pSRSapI-TS recombinant expression vector

[0059] 3. Construction of pSRSapI-TSM recombinant expression vector

[0060] To transform the N-terminus of the TS gene, it is necessary to insert a large R-based amino acid triplet sequence before and after the start codon through primer cloning, and reintegrate the gene and plasmid fragment through homologous recombination. Design two pairs of primers and clone them with the recombinant plasmid pSRSapI-TS as a template. The resulting vector and gene fragment for homologous recombination are named pSRSapI-if and TSM-if. The amplification primer sequences of the fragment pSRSapI-if are as follows: pSRSapI-if-F1 (SEQ ID NO: 5): 5'-GTACTCAAGCTCGTAACGAAGG-3'; pSRSapI-if-R1 (SEQ ID NO: 6): 5'-GATAGTTGTGCCAT CATGGATTTCTC-3'; Reaction system: KODOneTM PCR MasterMix-Blue-25μL, pSRSapI-if-F1 (10μM) 1.5μL, pSRSapI-if-R1 (10μM) 1.5μL, pSRSapI-TS 200ng, ddH 2 O up to 50μL; PCR amplification conditions are as follows: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 55℃ annealing for 30s, 68℃ extension for 3min, 35 cycles, 68℃ extension for 5min. The amplification primer sequences of fragment TSM-if are as follows: TSM-if-F1 (SEQ ID NO: 7): 5'-GAGAAATCCATGATGGCACAACTATC-3'; TSM-if-R1 (SEQ ID NO: 8): 5'-CCTTCGTTACGAGCTTGAGTAC-3'; Reaction system: KOD OneTM PCRMaster Mix-Blue-25μL, TSM-if-F1 (10μM) 1.5μL, TSM-if-R1 (10μM) 1.5μL, pSRSapI-TS 200ng, ddH 2 O up to 50μL; PCR amplification conditions are as follows: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ renaturation for 10s, 68℃ extension for 90s, 35 cycles, 68℃ extension for 3min. The vector and fragment carry 20bp homologous sequence and have been linearized. The fragments pSRSapI-if and TSM-if were integrated by homologous recombination. The reaction system: 5×In-Fusion HD Enzyme Premix 2μL, pSRSapI-if 2μL, TSM-if 1μL, ddH 2 O Up to 10μL. Incubate at 50℃ for 15min to obtain the pSRSapI-TSM recombinant expression vector.

[0061] Example 2

[0062] The three recombinant expression vectors obtained in the examples were respectively used to transform Chlamydomonas reinhardtii and screen positive transformants. The experimental steps are as follows:

[0063] (1) Soak the glass beads in 0.1M hydrochloric acid solution overnight. Wash twice with anhydrous ethanol and dry in a 70°C oven for 4 hours. Weigh 300 mg of the treated glass beads into 5 mL centrifuge tubes and wrap them in tin foil. Sterilize in a high-temperature steam sterilizer at 121°C for 20 minutes, remove and dry for later use.

[0064] (2) In TAP medium supplemented with 100 μg / mL spectinomycin, 5 × 105 cells / mL inoculated with Chlamydomonas reinhardtii CC5168 and cultured at room temperature in the dark for 4 to 5 days until the cell density reached 1 to 2 × 10 6 cells / mL. Centrifuge at 5000rpm for 10min and collect cells from 300mL of algal liquid.

[0065] (3) Wash the cells once with high salt medium HSM and resuspend them with an appropriate amount of HSM medium until the algal cell density is about 2×10 8 cells / mL. At the same time, 10 μg of expression plasmid was taken and digested with 3 μL Eco 147I endonuclease at 37°C overnight in 30 μL system to linearize the expression plasmid. The linearized expression plasmid was purified and recovered.

[0066] (4) Take 300 μL of algae solution and transfer it to a 5 mL centrifuge tube containing 300 mg of glass beads. Add 10 μg of linearized expression plasmid and vortex the centrifuge tube at maximum speed for 15 to 20 seconds.

[0067] (5) Spread the mixture in the centrifuge tube onto an HSM plate, dry it, and invert it to avoid light overnight. Then place the plate at 70 μmol / (m 2 ·s) Culture at 25°C under light for 4 to 6 weeks until green monoclonal transformants grow.

[0068] (6) Pick the green transformants and streak them on HSM medium. Place the plate at 70 μmol / (m 2 s) Cultivate under light until new colonies grow to accelerate the homogenization of chloroplast transformants. After the transformants grow, repeat the streaking once. Pick a single algae colony with rapid growth and use chelex to extract the total DNA of the transformants.

[0069] (7) Using total DNA as a template, PCR was used to detect the TS gene in the transformants and its homogeneity in the chloroplasts. The primer pair TSF1 (SEQ ID NO: 9): 5'-GGACAGGTATCCGGTAAGCG-3', TSR1 (SEQ ID NO: 10): 5'-CGTGTACGACCTGAACGTG-3' was used to detect whether the TS gene was present in the transformants. The PCR amplification conditions were as follows: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 50°C annealing for 10 s, 68°C extension for 10 s, and after 35 cycles, 68°C extension for 3 min. The primer pair F1 (SEQ ID NO: 11): 5'-GTCATTGCGAAAATACTGGTGC-3' and R1 (SEQ ID NO: 12): 5'-CGGATGTAACTCAATCGGTAG-3' was used to detect the homogeneity of the TS gene in the chloroplasts of the transformants. The PCR amplification conditions were as follows: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 50°C renaturation for 10 s, 68°C extension for 90 s, and 68°C extension for 3 min after 35 cycles. The PCR product of F1 / R1 was 0.88 kb before homogenization was completed, but no PCR product was found in F1 / R1 after homogenization was completed. Transformants with a PCR product size of 2.7 kb when amplified with primers TSF1 / TSR1 and no PCR product when amplified with primers F1 / R1 were selected as positive transformants.

[0070] In the pASapI-TS vector transformation experiment, a total of 4 strains with TS gene inserted and correctly integrated were screened out after verification, named A1, A2, A3 and A4 respectively; in the pSRSapI-TS vector transformation experiment, a total of 2 strains with TS gene inserted and correctly integrated were screened out after verification, named P1 and P2 respectively; in the pSRSapI-TSM vector transformation experiment, a total of 4 strains with TS gene inserted and correctly integrated were screened out after verification, named PM1, PM2, PM3 and PM4 respectively. The above strains were used for subsequent detection.

[0071] (8) Select positive transformants and inoculate them into fresh anti-TAP liquid culture medium at a concentration of 4%, and continue to culture under light for 2 to 6 days. Use a refrigerated centrifuge at 5000rpm, 4℃, centrifuge for 6min, collect 100mL of algae liquid cells, add appropriate amount of PBS to wash, resuspend once, and centrifuge. Add 1mL of Western lysis buffer containing 10μg / mL protease inhibitors to resuspend the cells, ice bath for 10min, and oscillate and resuspend once every 2min. Use an ultrasonic device to disrupt the cells, the processing procedure is: power 60W, on 2s, off 2s, and disruption for 5min. Centrifuge at 12000rpm, 4℃ for 30min, and take the supernatant into a clean 1.5mL centrifuge tube.

[0072] (9) Using mouse monoclonal antibody (HA-tag antibody, abcam) as the primary antibody, horseradish peroxidase-labeled goat anti-mouse IgG (HRP-labeled Goat Anti-Mouse IgG) as the secondary antibody, and RBP40 protein as the internal reference, Western Blot was used to detect the 3×HA tag carried on TS.

[0073] The present invention selects the total protein extract of Escherichia coli with the relevant target gene as the positive control, and selects the total protein extract of CC5168 wild-type algae and pASapI empty vector transformed algae as the negative control. The obtained protein expression results are shown as follows Figure 2 :The positive control had a clear band at 100 kDa, which was consistent with the expected size, while no corresponding band was observed in the negative control group (CC5168 and C1). Transformants A1, P1 and PM1 all produced protein bands around 100 kDa. This indicates that after the TS gene was recombinantly integrated into Chlamydomonas reinhardtii, the taxadiene synthase protein was successfully expressed. The protein accumulation results are shown in Figure 3 : There was almost no accumulation of taxadiene synthase protein in the negative control group (CC5168 and C1), while taxadiene synthase protein accumulated in the transformant strains A1, P1 and PM1, and the accumulation of taxadiene synthase protein was higher on the second day. The accumulation of taxadiene synthase protein in PM1 strain was significantly higher than that in A1 and P1 strains.

[0074] The present invention selects positive transformants that can be significantly positive in WB detection as engineered algae. The obtained algae strain A1 is regulated by atpA promoter and is recorded as engineered algae TSA (CCTCC M 20221695); the algae strain P1 obtained by screening is regulated by psaA promoter and is recorded as engineered algae TSP (CCTCC M20221697); the algae strain PM1 obtained by screening is regulated by psaA promoter and the 5' end of TS is optimized, and is recorded as engineered algae TSPM (CCTCC M 20221696).

[0075] Example 3

[0076] Taxadiene was produced using the engineered algae TSA, TSP and TSPM obtained in Example 2 respectively:

[0077] The engineered algae were cultured in a six-well plate with 4.5 mL of TAP medium containing 100 μg / mL carbenicillin, and 500 μL of dodecane was overlaid on the medium as a two-phase culture system to facilitate the collection and detection of taxadiene. The inoculation density of the engineered algae was about 7×10 6cells / mL, centrifuged at 5000rpm for 8min to precipitate algal cells, removed the supernatant of the inoculation solution and resuspended in the two-phase culture system. After inoculation, the six-well cell culture dish was placed in a constant temperature and light shaking incubator at 15℃ and 120rpm and heated at 70μmol / (m 2 ·s) light intensity, and culture under continuous light for 8 days.

[0078] The dodecane phase was collected and centrifuged at 12000 rpm for 2 min. The clarified dodecane was aspirated and filtered through a 0.22 μm nylon 66 membrane. The filtrate was transferred to a gas chromatography bottle. The product was detected by GC-MS. The results are shown in Figure 4 . Figure 4 The results showed that among the three engineered algae, the taxadiene synthase translated by TS had biocatalytic activity in the chloroplasts of Chlamydomonas reinhardtii and could successfully catalyze GGPP into taxadiene.

[0079] The product peaks of each algae strain were normalized, and the productivity index was calculated based on the physiological data. The volume yield of the TSPM algae strain was 26.7±1.3μg / L, and the cell yield was 2.2±0.3fg / cell. The volume yield of the TSA algae strain was 1.9±0.1μg / L, and the cell yield was 0.1±0.005fg / cell. The volume yield of the TSP algae strain was 10.7±0.4μg / L, and the cell yield was 0.6±0.02fg / cell.

[0080] Example 4

[0081] The difference between this embodiment and embodiment 3 is that: based on the acetate concentration in TAP, 4 times of acetate is additionally added to the culture medium to prepare the TAP culture medium.

[0082] Example 5

[0083] The difference between this embodiment and embodiment 4 is that 0.5 mM methyl jasmonate is added to the culture medium.

[0084] Example 6

[0085] This example uses the TSPM algae strain to conduct group control experiments on the amount of acetate added, inoculation density, lighting conditions and culture temperature (the other conditions not mentioned are the same as those in Example 3):

[0086] (1) Control experiment 1: A series of TAP medium were prepared by doubling the amount of Tris-Acetate mother solution, and named T(N)AP according to the multiple used. HSM medium, TAP medium or doubled acetate medium TNAP (N=1-7) were selected as the culture medium for two-phase fermentation control. The light intensity was 70 μmol / (m 2·s), and culture control was performed based on two lighting modes: continuous light and light-dark alternation (L:D=16h:8h).

[0087] (2) Control experiment 2: A series of TAP medium were prepared in the form of doubling the amount of Tris-Acetate mother solution, and named T(N)AP according to the multiple used. HSM medium, TAP medium or doubled acetate medium TNAP (N=1-7) were selected as the culture medium for dual-phase fermentation control. The culture temperature was set at 15°C, 25°C, and 30°C for culture control.

[0088] (3) Control experiment 3: A series of TAP medium were prepared in the form of doubling the amount of Tris-Acetate mother solution, and named T(N)AP according to the multiple of use. HSM medium, TAP medium or doubled acetate medium TNAP (N=1-7) were selected as the medium for two-phase fermentation control. The inoculation density was set to low density (5×10 5 cells / mL), high density (7×10 6 cells / mL) and high density (7×10 6 cells / mL)+0.5 mM methyl jasmonate was used as a culture control.

[0089] Taxadiene was collected under different experimental conditions, and the results are shown in Figure 5 .

[0090] Depend on Figure 5 a It can be seen that under the two lighting modes, the taxadiene production of the transformant PM1 in acetate culture was higher than that in carbon-deficient autotrophic fermentation, and the optimal culture medium was T6AP. In addition, the maximum yield was lower when the light-dark alternating culture was used, with a volumetric yield of 32.3±0.9μg / L, which was about 61.3% of that in continuous light culture.

[0091] Depend on Figure 5 b It can be seen that the transformant PM1 had the highest yield when cultured at 15°C, followed by 25°C, and the lowest yield when cultured at 30°C. When cultured at 15°C, the best medium was TAP, with the highest volumetric yield of 69.2±2.9μg / L and the highest cell yield of 6.5±0.3fg / cell (n=3).

[0092] Depend on Figure 5 c It can be seen that when the transformant PM1 was inoculated at high density, the optimal medium was T3AP, the acetate concentration was higher than that of normal inoculation, and the volume yield was 146.4±2.1μg / L. After adding methyl jasmonate, the optimal medium was still T3AP, and the volume yield was 185.5±4.4μg / L.

[0093] Example 7

[0094] The engineered algae TSPM obtained in Example 2 was used to produce taxadiene:

[0095] The engineered algae were cultured in 4.5 mL of TAP medium containing 100 μg / mL carbenicillin in a six-well plate, and 500 μL of dodecane was overlaid on the medium as a two-phase culture system to facilitate the collection and detection of taxadiene (2 times of acetate was added to the medium, and 0.5 mM methyl jasmonate was added as an exogenous stimulus). The inoculation density of the engineered algae was about 7×10 6 cells / mL, centrifuged at 5000rpm for 8min to precipitate algal cells, removed the supernatant of the inoculation solution and resuspended in the two-phase culture system. After inoculation, the six-well cell culture dish was placed in a constant temperature and light shaking incubator at 15℃ and 120rpm and heated at 70μmol / (m 2 ·s) light intensity, continuous light cultivation, and collection of products every day, the results are shown in Figure 6 .

[0096] Depend on Figure 6 It can be seen that during the entire culture process, the cell density continued to increase, and a slight decrease occurred on day 8. The accumulation level of taxadiene showed an S-shaped growth, with a volume yield of 185.5±4.4 μg / L and a unit cell yield of 5.6±0.2 fg / cell (n=3) on day 8.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a taxadiene synthase gene heterologously expressed in chloroplasts of Chlamydomonas reinhardtii in the construction of a recombinant strain of Chlamydomonas reinhardtii, characterized in that: The nucleotide sequence of the gene is SEQ ID NO:

1.

2. A recombinant algae strain, characterized in that: The recombinant algae strain is selected from any one of the following: (1) Recombinant algae strain TSA (Chlamydomonas reinhardtii TAS), deposited with CCTCC NO: M20221695, deposited with China Center for Type Culture Collection, and deposited on October 31, 2022; (2) Recombinant algae strain TSPM (Chlamydomonas reinhardtii TSPM), deposit number is CCTCC NO: M20221696, deposited by China Center for Type Culture Collection, deposit date is October 31, 2022; (3) Recombinant algal strain TSP (Chlamydomonas reinhardtii TSP), deposit number is CCTCC NO: M20221697, deposited by China Center for Type Culture Collection, and deposited on October 31, 2022.

3. A method for producing taxadiene, characterized in that: The recombinant algae strain according to claim 2 is used as the production algae strain, and dodecane is added to TAP medium for biphasic culture to obtain taxadiene.

4. The method according to claim 3, characterized in that The culture conditions are continuous illumination, the culture temperature is 15-30°C, and the inoculation density of the recombinant algae strain is 1×10 6 ~10×10 6 cells / mL.

5. The method according to claim 3, characterized in that: Acetate and / or methyl jasmonate are added to the TAP medium.

6. Use of the recombinant algae strain according to claim 2 or the method for producing taxadiene according to any one of claims 3 to 5 in the preparation of taxadiene.

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