Engineering algal strain capable of synthesizing alpha-farnesene and preparation method thereof

Abstract

The invention relates to the field of genetic engineering and bioengineering, in particular to engineering algae capable of synthesizing alpha-farnesene and a preparation method of the engineering algae. An MEP metabolic network of synechocystis PCC 6803 is modified, through single-copy series recombination and multi-copy combination of dxs, ispA, idi and AFS genes, optimization of a farnesene MEP metabolic pathway is achieved, the carbon flux of oriented synthesis of farnesene is precisely regulated and controlled, under the optimal effect, the yield of alpha-farnesene within 9 days is 83.07 mg / L (25.82 mg / g DCW), and economic application value is achieved.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and bioengineering, specifically to engineered algae capable of synthesizing α-farnesene and their preparation methods. Background Technology

[0002] Farnesene (3,7,11-trimethyl-1,3,6,10-dodecathatene; C 15 H 24 Farneseene, also known as farnesene, is an acyclic volatile sesquiterpene first discovered in apple peels and is mainly found in essential oils such as sweet orange oil, rose oil, ylang-ylang oil, and mandarin orange oil. It has significant value in the fields of medicine (nutritional supplements), cosmetics, and bioenergy. For example, farnesene can be used to prepare high-calorific-value, efficient, clean, and renewable fuels, a novel fuel that is an ideal alternative to blended aviation fuels, thus attracting considerable attention. Farnesene is an important raw material in the pharmaceutical field, used to synthesize isophytol, a precursor to vitamin E, one of the world's largest vitamin products. Its production process reduces carbon emissions by 60%, making it more environmentally friendly than traditional chemical synthesis techniques. In the chemical industry, farnesene can also be used as an additive to improve the plasticity of rubber; these new farnesene-based materials exhibit different properties, such as reduced rolling resistance, improved compression and permanent fixation, and greater softness. Furthermore, farnesene can be used in the production of lubricants, surfactants, and cosmetics.

[0003] Extracting farnesene from plants for mass market use is challenging due to the relatively low content of farnesene in plants and the significant impact of seasonal and regional climate factors on plant growth. Research on the chemical synthesis of farnesene faces unavoidable problems such as material availability, high production costs, low production efficiency, and environmental pollution. Microbial synthesis, with its short growth cycle, minimal environmental impact, and lack of arable land occupation, overcomes the shortcomings of natural extraction and chemical synthesis, making it an ideal choice for farnesene production. α-Farnesene has been synthesized in various microorganisms, including yeast, Escherichia coli, and cyanobacteria, through metabolic engineering strategies. However, yeast and E. coli, as heterotrophic organisms, rely on expensive organic carbon sources, which diminishes the economic and environmental benefits of the process. Cyanobacteria, on the other hand, have significant advantages over plants in terms of photosynthetic efficiency (above 10%) and growth rate. Furthermore, utilizing photosynthetic microbial cell factories for the biosynthesis of biofuels and chemicals offers better stereoselectivity, milder reaction processes, and lower toxicity compared to traditional chemical synthesis. To date, some progress has been made in the preparation of farnesene using cyanobacteria as host cells. The application of cyanobacteria can effectively utilize carbon dioxide to realize a "light-driven cell factory" for the production of green synthetic chemicals, while alleviating urgent environmental and energy pressures, which is of great significance.

[0004] Cyanobacteria are ideal cellular factories for carbon-negative chemical production, possessing immense potential to directly utilize light and carbon dioxide as their sole energy and carbon sources. The metabolic pathways of terpenes are long and complex, with poor enzyme specificity, low selectivity, and low efficiency, posing challenges to their synthesis in heterologous cellular factories. Currently, efficient and targeted synthesis of various terpenes has been achieved in different cyanobacterial chassis, fully demonstrating the feasibility of terpene synthesis using cyanobacteria as a chassis. Synechococcus pyrenoids PCC6803, Synechococcus polymorpha PCC7942, and Synechococcus polymorpha PCC7002 have become very promising hosts for terpene production. However, the reported engineered algae have not achieved ideal yields of farnesene. In cyanobacteria, farnesene is synthesized via the methyl erythritol-phosphate (MEP) pathway, using glyceraldehyde-3-phosphate and pyruvate produced during photosynthesis as substrates, converting them into the common precursors of all terpenes, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP). For farnesene synthesis, two molecules of IPP and one molecule of DMAPP can generate farnesylpyrophosphate (FPP). Farnesene can be produced by overexpressing farnesene synthase using FPP as a substrate. Although metabolic strategies have seen incremental improvements, the titers obtained in engineered cyanobacteria strains (e.g., ~2.22 mg / L / d) have remained low. (Sun, J., Xu, X., Wu, Y., Sun, H., Luan, G., Lu, X. 2023. Conversion of carbon dioxide into valencene and other sesquiterpenes with metabolic engineered...) Synechocystis (sp. PCC 6803 cell factories. GCB Bioenergy, 15(9), 1154-1165) is still insufficient to achieve industrial feasibility.

[0005] Therefore, it is necessary to develop an engineered algae that can synthesize α-farnesene in high yield to overcome the above technical problems. Summary of the Invention

[0006] The purpose of this invention is to address the problems of low yield, significant metabolic bottlenecks, and limited industrialization prospects in the synthesis of α-farnesene by cyanobacteria in existing technologies. By constructing a multi-copy key enzyme gene expression system and optimizing carbon flux allocation in the MEP metabolic pathway, efficient synthesis of α-farnesene can be achieved.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an engineered algal strain capable of synthesizing α-farnesene, wherein the engineered algal strain is Synechocystis (… Synechocystissp. PCC 6803 is a chaste alga, and the algal strain includes modified plasmids of several key enzyme gene expression frames to enhance the metabolic flux of the MEP pathway and promote the synthesis and accumulation of α-farnesene. The key enzyme genes include dxs (1-Deoxy-D-xylitol-5-phosphate synthase) idi (Isopentene pyrophosphate isomerase) ispA (Farnesy pyrophosphate synthase) AFS (α-Farnese synthase), the above genes have undergone codon optimization for cyanobacterial sequences, as shown in Seq No. 1~4; The key enzyme gene expression cassette is constructed by constructing the above-mentioned key enzyme genes individually or in combination; The enzyme gene expression cassette is tandemly integrated into one or more of the neutral sites Slr0168, Slr9394, and Slr1556. The enzyme gene expression cassette is driven by one or more of the promoters PcpcB560 and PrbcL6803.

[0008] Preferably, the engineered algal strain employs a multi-copy tandem strategy, with key enzyme genes repeatedly appearing at different neutral sites.

[0009] Preferably, the enzyme gene is inserted between upstream and downstream homologous arms at the neutral site.

[0010] Preferably, the combination of the key enzyme gene expression cassettes includes idi - ispA, idi - ispA - dxs, AFS - ispA, AFS - idi, AFS - dxs, AFS - ispA - idi, AFS - ispA - dxs, AFS - idi - dxs, AFS - ispA - idi - dxs .

[0011] Preferably, in the key enzyme gene expression frame, PrbcL6803 drives dxs and combinations containing dxs; PcpcB560 drives the remaining genes.

[0012] Preferably, the engineered algal strain is: Slr0168 site insertion. AFS - ispA Slr1556 site insertion AFS - ispA Slr9394 site insertion AFS - ispA - idi - dxs .

[0013] Secondly, the present invention provides a method for preparing the engineered algal strain described in the first aspect, comprising the following steps: S1: Gene for key enzyme synthesis AFS, ispA, idi, dxs ; S2: Construct an expression cassette containing the above genes, insert one or more of the neutral sites Slr0168, Slr9394, and Slr1556, and ligate it with a plasmid vector to form a modified plasmid. S3: The modified plasmid will be transformed, subjected to resistance screening, and validated by PCR to obtain a stably integrated engineered algal strain.

[0014] Preferably, the plasmid vector is pUC19.

[0015] Thirdly, the present invention provides an application of the engineered algal strain described in the first aspect in the synthesis of α-farnesene.

[0016] Preferably, the algal strain utilizes CO2 as a carbon source for the photosynthetic synthesis of α-farnesene under light conditions.

[0017] The concept of this invention is: To increase the yield of α-farnesene in cyanobacteria, metabolic engineering efforts have primarily focused on overcoming bottlenecks in the MEP pathway. In this section, AFS The production of α-farnesene is determined by factors such as activity, intracellular IPP / DMAPP levels, and FPP pool capacity. This typically involves overexpression of key enzymes, such as 1-deoxy-d-xyulose-5-phosphate synthase (DAP). dxs ), isopentenyl diphosphate isomerase ( idi ) and farnesyl diphosphate synthase ( ispA ), while optimizing AFS Expression. A significant limitation of traditional single-gene overexpression is its inability to fully address complex metabolic bottlenecks. Furthermore, this modification often increases cellular burden, potentially limiting further yield increases; increasing the copy number of key pathway genes is a promising strategy to enhance enzyme expression levels and metabolic flux by providing more transcriptional templates.

[0018] The beneficial effects of this invention are: This invention provides a method for developing and implementing multicopy metabolic engineering in Synechocystis, modifying the MEP metabolic network of Synechocystis PCC6803, through... dxs, ispA, idi and AFS Single-copy tandem recombination and multi-copy combination of genes were used to optimize the farnesene MEP metabolic pathway by "increasing manpower to compete for resources," precisely regulating the carbon flux directed to the synthesis of farnesene, resulting in QZ21 ( AFS - ispA - idi - dxs - AFS - ispA - AFS - ispA The yield of α-farnesene was 83.07 mg / L (25.82 mg / g DCW) within 9 days. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid pQZ1.

[0020] Figure 2 This is a schematic diagram of the structures of recombinant plasmids pQZ2, pQZ3, pQZ4, pQZ5, and pQZ6.

[0021] Figure 3 This is a schematic diagram of the structures of recombinant plasmids pQZ12, pQZ13, pQZ14, pQZ15, pQZ16, pQZ17, pQZ18, and pQZ19.

[0022] Figure 4 This is a chromatogram of farnesene detected by GC-MS.

[0023] Figure 5 This is a mass spectrum of farnesene detected by GC-MS.

[0024] Figure 6 These are electrophoretic images of the amplification products of WT and engineered algal strains QZ1-QZ5 at neutral sites Slr0168 and Slr9394.

[0025] Figure 7 This is a graph showing the α-farnesene production of engineered algal strains QZ1-QZ5 over 9 days.

[0026] Figure 8 This is a graph showing the proportion of α-farnesene accumulation in QZ1-QZ5 cells to cell dry weight.

[0027] Figure 9 These are electrophoretic images of the amplification products of engineered algal strain QZ6-QZ24 at neutral sites Slr0168, Slr1556, and Slr9394.

[0028] Figure 10 This is a graph showing the α-farnesene production of engineered algal strains QZ5-QZ18 over 9 days.

[0029] Figure 11 This is a graph showing the percentage of α-farnesene accumulation in the dry weight of engineered algal strains QZ5-QZ18 over 9 days.

[0030] Figure 12 This is a graph showing the α-farnesene production of engineered algal strains QZ12 and QZ19-QZ21 over 9 days.

[0031] Figure 13 This is a graph showing the percentage of α-farnesene accumulation in the dry weight of engineered algal strains QZ12 and QZ19-QZ21 over 9 days. Detailed Implementation

[0032] Example 1 1. Algal strains used The basal alga used in this invention is Synechocystis PCC6803, which is often used as a model strain in related experimental studies. 2. Plasmid construction: The genes involved include: E. coli FPP synthase of MG1655 ispAGene (farnesyl pyrophosphate synthase, NP_414955) dxs Gene (1-deoxy-D-xylitol-5-phosphate, NP_414954). S. cerevisiae The idi gene (isopentene pyrophosphate isomerase, NP_015208) of BY4741; derived from... Malus x domestica The α-farnesene synthase (AY182241) was used. The above genes underwent codon optimization targeting cyanobacterial sequences, as shown in Seq Nos. 1-4. Multi-copy integration vectors were constructed at neutral sites Slr9394, Slr0168, and Slr1556 to integrate key genes of the MEP metabolic pathway.

[0033] 1) Farnese synthase ( AFS The coding frames of the enzyme were connected downstream of the promoter PcpcB560 to form the synthase expression frame. Then, they were inserted between the upstream and downstream homologous arms of the neutral sites Slr0168 and Slr1556 and inserted into the pUC19 backbone. Spectinomycin / chloramphenicol resistance was used as a selection marker to form recombinant plasmids pQZ1 and pQZ12.

[0034] 2) Genes idi - ispA It is connected in series downstream of the promoter PcpcB560 to form idi - ispA Expression frames, which contain genes dxs The fragment is connected downstream of the promoter PrbcL6803 to form a structure. dxs Expression boxes, concatenating two expression boxes together to obtain idi - ispA - dxs Expression box, then ispA Expression box / idi Expression box / idi - ispA Expression box / idi - ispA - dxs Expression frames were inserted between the upstream and downstream homologous arms of the neutral site Slr9394, respectively. Using kanamycin resistance as a selection marker, the above fragments were inserted into the pUC19 backbone to obtain recombinant plasmids pQZ2, pQZ3, pQZ4, and pQZ5, respectively.

[0035] 3) Genes AFS - ispA It is connected in series downstream of the promoter PcpcB560 to form AFS - ispA Expression frames, which contain genes dxs - idi The fragment is connected downstream of the promoter PrbcL6803 to form a structure. dxs - idi Expression boxes, concatenating two expression boxes together to obtain AFS - ispA - dxs - idiThe expression frames were then inserted between the upstream and downstream homologous arms of the neutral sites Slr9394, Slr1556 and Slr0168, respectively, and inserted into the pUC19 backbone. Using kanamycin / chloramphenicol / spectinomycin resistance as a selection marker, recombinant plasmids pQZ6, pQZ19 and pQZ11 were constructed.

[0036] 4) Genes AFS - ispA It is connected in series downstream of the promoter PcpcB560 to form AFS - ispA The expression frame was then inserted between the upstream and downstream homologous arms of the neutral sites Slr0168 and Slr1556, and then inserted into the pUC19 backbone. Spectinomycin / chloramphenicol resistance was used as a selection marker to construct recombinant plasmids pQZ7 and pQZ13.

[0037] 5) Genes AFS - idi It is connected in series downstream of the promoter PcpcB560 to form AFS - idi The expression frame was then inserted between the upstream and downstream homologous arms of the neutral sites Slr0168 and Slr1556, and then inserted into the pUC19 backbone. Spectinomycin / chloramphenicol resistance was used as a selection marker to construct recombinant plasmids pQZ8 and pQZ14.

[0038] 6) Genes AFS Downstream of the promoter PcpcB560 AFS Expression Frames, Genes dxs The fragment is connected downstream of the promoter PrbcL6803 to form a structure. dxs Expression boxes, concatenating two expression boxes together to obtain AFS - dxs The expression frame was then inserted between the upstream and downstream homologous arms of the neutral sites Slr0168 and Slr1556, and then inserted into the pUC19 backbone. Spectinomycin / chloramphenicol resistance was used as a selection marker to construct the recombinant plasmids pQZ9 and pQZ16.

[0039] 7) Genes AFS - ispA It is connected in series downstream of the promoter PcpcB560 to form AFS - ispA Expression Frames, Genes idi The fragment is connected downstream of the promoter PrbcL6803 to form a structure. idi Expression boxes, concatenating two expression boxes together to obtain AFS - ispA - idi The expression frame was then inserted between the upstream and downstream homologous arms of the neutral sites Slr0168 and Slr1556, and then inserted into the pUC19 backbone. Spectinomycin / chloramphenicol resistance was used as a selection marker to construct recombinant plasmids pQZ10 and pQZ15.

[0040] 8) Genes AFS - ispA It is connected in series downstream of the promoter PcpcB560 to formAFS - ispA Expression Frames, Genes dxs The fragment is connected downstream of the promoter PrbcL6803 to form a structure. dxs Expression boxes, concatenating two expression boxes together to obtain AFS - ispA - dxs The expression frame was then inserted between the upstream and downstream homologous arms of the neutral site Slr1556 and onto the pUC19 backbone, using chloramphenicol resistance as a selection marker to form the recombinant plasmid pQZ17.

[0041] 9) Genes AFS Downstream of the promoter PcpcB560 AFS Expression frames, which contain genes dxs - idi The fragment is connected downstream of the promoter PrbcL6803 to form a structure. dxs - idi Expression boxes, concatenating two expression boxes together to obtain AFS - dxs - idi The expression frame was then inserted between the upstream and downstream homologous arms of the neutral site Slr1556 and onto the pUC19 backbone, using kanamycin / chloramphenicol resistance as a selection marker to form the recombinant plasmid pQZ18.

[0042] 3. Construction of engineered algal strains: 1) Construction of algal strains QZ1, QZ22, and QZ24 Plasmids pQZ1, pQZ6, and pQZ5 were introduced into wild-type *Syntrophus synergae* PCC6803 to obtain algal strains QZ1, QZ22, and QZ24, respectively. The method is as follows: 1 mL of wild-type *Syntrophus synergae* PCC6803 in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmids pQZ1, pQZ6, and pQZ5 were added respectively and mixed thoroughly (plasmid concentration 200 μg / mL). The mixture was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at a controlled temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing the corresponding resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto a BG11 solid plate containing the same resistance for colony enrichment. After the algal strains grow, perform genotyping; through screening, completely isolated algal strains QZ1, QZ22, and QZ24 are obtained.

[0043] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 6 , 9 As shown, the Slr0168 site of algal strain QZ1 was inserted with AFSSynthase expression cassette; the Slr9394 site of QZ22 was inserted. AFS - IspA - Dxs - Idi Gene expression cassette; the Slr9394 site of QZ24 was inserted. Idi - IspA - Dxs Gene expression cassette.

[0044] 2) Construction of algal strains QZ2, QZ3, QZ4, and QZ5 Plasmids pQZ2, pQZ3, pQZ4, and pQZ5 were transferred into algal strain QZ1, respectively, to obtain algal strains QZ2, QZ3, QZ4, and QZ5. The method is as follows: 1 mL of QZ1 algal culture in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmids pQZ2, pQZ3, pQZ4, and pQZ5 were added and thoroughly mixed (plasmid concentration 200 μg / mL). The mixture was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at room temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing spectinomycin / kanamycin resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto BG11 solid plates containing the same resistance for colony enrichment. After the algal strains grow, perform genotyping; through screening, completely isolated algal strains QZ2, QZ3, QZ4, and QZ5 are obtained.

[0045] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 6 As shown, the Slr9394 site of algal strains QZ2, QZ3, QZ4, and QZ5 was inserted with [insert insertion site here]. IspA Expression box / Idi Expression box / Idi - IspA Expression box and Idi - IspA - Dxs Expression frames; Slr0168 sites were inserted in algal strains QZ2, QZ3, QZ4, and QZ5. AFS Synthase expression cassette.

[0046] 3) Construction of algal strains QZ6, QZ7, QZ8, QZ9, and QZ10 Algal strain QZ24 was transformed with plasmids pQZ7, pQZ8, pQZ9, pQZ10, and pQZ11, respectively, to obtain algal strains QZ6, QZ7, QZ8, QZ9, and QZ10. The method is as follows: 1 mL of QZ24 algal culture in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmids pQZ7, pQZ8, pQZ9, pQZ10, and pQZ11 were added and thoroughly mixed (plasmid concentration 200 μg / mL). The mixture was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at a controlled temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing spectinomycin / kanamycin resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto BG11 solid plates containing the same resistance for colony enrichment. After the algal strains grow, perform genotyping; through screening, completely isolated algal strains QZ6, QZ7, QZ8, QZ9, and QZ10 are obtained.

[0047] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 9 As shown, the Slr0168 site of algal strains QZ6, QZ7, QZ8, QZ9, and QZ10 was inserted with [insert insertion site here]. AFS - IspA Expression box / AFS - Idi Expression box / AFS - Dxs Expression box / AFS - Idi - IspA Expression box and AFS - Idi - IspA - Dxs Expression box.

[0048] 4) Construction of algal strains QZ11, QZ12, QZ13, QZ14, QZ15, QZ16, QZ17, and QZ18 Plasmids pQZ12, pQZ13, pQZ14, pQZ15, pQZ16, pQZ17, pQZ18, and pQZ19 were transferred into algal strain QZ5 to obtain algal strains QZ11, QZ12, QZ13, QZ14, QZ15, QZ16, QZ17, and QZ18. The method is as follows: Take 1 mL of QZ5 algal culture in the exponential growth phase into a sterile EP tube, centrifuge at 5000 g for 10 min and discard the supernatant. Resuspend the precipitate in 400 μL of antibiotic-free BG11 solution. Then add approximately 4 μg of plasmids pQZ12, pQZ13, pQZ14, pQZ15, pQZ16, pQZ17, pQZ18, and pQZ19 respectively and mix thoroughly (plasmid concentration is 200 μg / mL). Place in a constant temperature and light incubator (30℃, 70 μmol photons / mL).2 / s) Incubate at a controlled temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing spectinomycin / kana / chloramphenicol resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto BG11 solid plates containing the same resistance for colony enrichment. After the algal strains grow, perform genotyping; through screening, completely isolated algal strains QZ11, QZ12, QZ13, QZ14, QZ15, QZ16, QZ17, and QZ18 are obtained.

[0049] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 9 As shown, the Slr1556 site of algal strains QZ11, QZ12, QZ13, QZ14, QZ15, QZ16, QZ17, and QZ18 was inserted with [insert insertion site here]. AFS Expression box / AFS - IspA Expression box / AFS - Idi Expression box / AFS - Dxs Expression box / AFS - IspA - Dxs Expression box / AFS - Idi - IspA Expression box / AFS - Idi - Dxs Expression box and AFS - Idi - IspA - Dxs Expression box.

[0050] 5) Construction of algal strain QZ19 Algal strain QZ6 was transformed with plasmid pQZ13 to obtain algal strain QZ19. The method is as follows: 1 mL of QZ6 algal culture in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmid pQZ13 was added and thoroughly mixed (plasmid concentration 200 μg / mL). The tube was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at room temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing spectinomycin / kana / chloramphenicol resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto BG11 solid plates containing the same resistance for colony enrichment. After the algal strain grows, perform genotyping; the completely isolated algal strain QZ19 is obtained through screening.

[0051] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 9 As shown, the Slr1556 site of algal strain QZ19 was inserted with [insert insertion sites here]. AFS - IspA Expression frames; Slr0168 sites were respectively insertedAFS - IspA Expression box; Slr9394 site was inserted Idi - IspA - Dxs Gene expression cassette.

[0052] 6) Construction of algal strain QZ23 Plasmid pQZ13 was transferred into algal strain QZ22 to obtain algal strain QZ23. The method is as follows: 1 mL of QZ22 algal culture in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmids pQZ1 and pQZ7 were added separately and mixed thoroughly (plasmid concentration 200 μg / mL). The mixture was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at a controlled temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing kanamycin / chlorine resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto a BG11 solid plate containing the same resistance for colony enrichment. After the algal strain grows, perform genotyping; the completely isolated algal strain QZ23 is obtained through screening.

[0053] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 9 As shown, the Slr01556 site of algal strain QZ23 was inserted with [insert insertion sites here]. AFS - IspA Expression box.

[0054] 7) Construction of algal strains QZ20 and QZ21 Algal strains QZ23 were transformed with plasmids pQZ1 and pQZ7, respectively, to obtain algal strains QZ20 and QZ21. The method is as follows: 1 mL of QZ23 algal culture in the exponential growth phase was transferred to a sterile EP tube, centrifuged at 5000 g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μL of antibiotic-free BG11 solution. Then, approximately 4 μg of plasmids pQZ1 and pQZ7 were added and thoroughly mixed (plasmid concentration 200 μg / mL). The mixture was then placed in a constant temperature and light incubator (30℃, 70 μmol photons / mL). 2 / s) Incubate at a controlled temperature for 24 h. Spread the treated algal solution onto a nitrocellulose membrane (placed on an antibiotic-free BG11 solid plate) and incubate in a constant temperature and light incubator for 24 h; then transfer the nitrocellulose membrane to a BG11 plate containing spectinomyces / kana / chlorine resistance and continue static incubation. After transformants grow in about 7 days, single colonies are picked and streaked onto BG11 solid plates containing the same resistance for colony enrichment. After the algal strains grow, perform genotyping; through screening, obtain the completely isolated algal strains QZ20 and QZ21.

[0055] The above algal strains were subjected to PCR detection, and the results are as follows: Figure 9 As shown, the Slr0168 site of algal strains QZ20 and QZ21 was inserted with [insert insertion site here]. AFS Expression box / AFS - IspA Expression box.

[0056] 4. Liquid culture of Synechocystis PCC 6803 and genetically engineered algal strains Using a sterile pipette tip, scrape bacterial cells from the plate into a 50 mL Erlenmeyer flask containing 25 mL of BG11 medium (with antibiotics), and place it on a constant temperature and light shaker (30℃, 70 μmol photons / m²). 2 After shaking and culturing at 150 rpm for about one week, it can be expanded into a 250 mL Erlenmeyer flask containing 200 mL of BG11 solution (with antibiotics). Air is then pumped into the Erlenmeyer flask to expand the culture (30℃, 150 μmol photons / m²). 2 / s). The OD of the above seed solution... 730 When the OD reaches 1.5–2.5, collect algal cells by centrifugation at 5000 g, resuspend the precipitate in fresh BG11 liquid medium, and calculate the OD required for column loading. 730 The algal cells were then inoculated into a column reactor (100 mL) for evaluation. In the formal evaluation experiment, the algal culture volume was 50 mL, and 8 mM TES-NaOH buffer (pH=8.0) was added to the column before loading. 10% (v / v) dodecane (CAS: 112-40-3) was added to the surface for product collection. Unless otherwise specified, the culture conditions for the light-driven carbon fixation and farnesene synthesis engineered algal strain were 30℃ and 100 µmol photons / m³. 2 / s, a mixed gas (3% CO2 + 97% air) is introduced. A set of samples is collected every 3 days for quantitative analysis. During the culture process, dodecane is replaced each time a sample is taken to ensure recovery efficiency.

[0057] 5. Identification and Quantitative Analysis of α-Farnese α-Farnesne samples produced by cyanobacterial strains in a photobioreactor were analyzed using GC-MS. The GC-MS parameters were set as follows: a TG-5 SILMS column (30 m × 250 µm inner diameter × 0.25 µm film thickness) was used. High-purity helium was used as the carrier gas, with a constant flow rate of 3 mL / min. The sample injection volume was 1 μL, the split ratio was 1:1, and the column oven temperature was initially maintained at 50 °C for 1 min, then increased to 100 °C at a rate of 50 °C / min and held at 100 °C for 1 min, then increased to 280 °C at a rate of 20 °C / min and held at 280 °C for 2 min. The total run time was 20 min, and the solvent delay time was set to 6 min. Mass spectrometry was performed in EI mode. The configuration was determined based on the NIST mass spectrometry library. Different concentrations of farnesene standards were prepared using ethyl acetate containing 5 mg / L caryophyllene oxide (Toronto Research Chemicals, >95%, C184730) as an internal standard, and the elution times were determined. Quantification was performed by the peak area ratio of farnesene to the internal standard caryophyllene oxide. A standard curve was plotted using gradient farnesene concentrations and the obtained peak area ratios. Previous experiments showed that the values ​​of the same sample after multiple GC-MS measurements were relatively similar. All α-farnesene standards used in this section were analytical grade (Toronto Research Chemicals, >90%, F102425). When quantifying with α-farnesene standards, the α-farnesene sample eluted at 7.86 min, and the internal standard caryophyllene oxide eluted at 8.50 min. Since the algal product was concentrated and collected using 10% (v / v) dodecane, the measured values ​​were divided by ten to represent the true production level of the algal strain. The results are shown in the attached figure.

Claims

1. An engineered algal strain capable of synthesizing α-farnesene, characterized in that, The engineered algal strain uses Synechocystis as the substrate algae, and the algal strain includes modified plasmids of several key enzyme gene expression frames to enhance the metabolic flux of the MEP pathway and promote the synthesis and accumulation of α-farnesene. The key enzyme genes include dxs (1-Deoxy-D-xylitol-5-phosphate synthase) idi (Isopentene pyrophosphate isomerase) ispA (Farnesy pyrophosphate synthase) AFS (α-Farnese synthase), as shown in Seq No. 1~4; The key enzyme gene expression cassette is constructed by constructing the above-mentioned key enzyme genes individually or in combination; The enzyme gene expression cassette is tandemly integrated into one or more of the neutral sites Slr0168, Slr9394, and Slr1556. The enzyme gene expression cassette is driven by one or more of the promoters PcpcB560 and PrbcL6803.

2. The engineered algal strain capable of synthesizing α-farnesene according to claim 1, characterized in that, The engineered algal strain employs a multi-copy tandem strategy, with key enzyme genes repeatedly appearing at different neutral sites.

3. The engineered algal strain capable of synthesizing α-farnesene according to claim 1, characterized in that, The enzyme gene is inserted between the upstream and downstream homologous arms of the neutral site.

4. The engineered algal strain capable of synthesizing α-farnesene according to claim 1, characterized in that, The combination of the key enzyme gene expression cassettes includes idi-ispA , idi-ispA-dxs , AFS-ispA , AFS-idi , AFS-dxs , AFS-ispA-idi , AFS-ispA-dxs , AFS-idi-dxs , AFS-ispA-idi-dxs .

5. The engineered algal strain capable of synthesizing α-farnesene according to claim 1, characterized in that, In the key enzyme gene expression cassette, PrbcL6803 drives... dxs and containing dxs The combination of genes; PcpcB560 drives the remaining genes.

6. The engineered algal strain capable of synthesizing α-farnesene according to claim 1, characterized in that, The engineered algal strain is: Slr0168 insertion site. AFS-ispA Slr1556 site insertion AFS-ispA Slr9394 site insertion AFS-ispA- idi-dxs .

7. A method for preparing the engineered algal strain according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Gene for key enzyme synthesis AFS, ispA, idi, dxs ; S2: Construct an expression cassette containing the above genes, insert one or more of the neutral sites Slr0168, Slr9394, and Slr1556, and ligate it with a plasmid vector to form a modified plasmid. S3: Transform the modified plasmid, screen for resistance, and verify with PCR to obtain a stable integrated engineered algal strain.

8. The method for preparing the engineered algal strain according to claim 7, characterized in that, The plasmid vector is pUC19.

9. The use of any one of the engineered algal strains according to claims 1 to 6 in the synthesis of α-farnesene.

10. The application of the engineered algal strain according to claim 9 in the synthesis of α-farnesene, characterized in that, The algal strain used CO2 as a carbon source to perform photosynthetic synthesis of α-farnesene under light conditions.

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