Genetic engineering construction and application of gibberellin GA4+7 efficient production strain

Abstract

The invention provides genetic engineering construction and application of a gibberellin GA4 + 7 efficient production strain. According to the method, the speed limiting step of a metabolic pathway of gibberella zeylanica is explored, key genes fpps, gs2, cps / ks and thmgr of the gibberella zeylanica are subjected to editing expression, the screened key genes ggs2, cps / ks and thmgr are subjected to co-expression, screening markers are recycled by using an inducible recombinase-based gibberella zeylanica screening marker recycling system, and finally, the strain OE: thmgr-gs2-cps / ks with the highest yield is obtained. The yield of GA4 + 7 in an optimized fermentation culture medium reaches 3.6 g / L.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to the preparation of gibberellin by bio-fermentation, and more specifically relating to a gibberellin GA. 4+7 Genetic Engineering Construction and Application of High-Efficiency Production Strains Background Technology

[0002] Gibberellins, as growth regulators, participate in multiple biological processes of plant growth and development. Compared with GA3, GA... 4+7 It has a more direct effect under certain specific needs, a wider range of applications, and milder operating conditions. In Fusarium oxysporum, due to the production of GA... 4+7 Catalyzed by cytochrome P450-3 oxidase, GA3, the final product of gibberellin, is eventually produced. Therefore, under natural conditions, GA3... 4+7 It is difficult to accumulate, resulting in high production costs and limited use.

[0003] Gibberellin, a classic secondary metabolite, is synthesized by *Gibberella fuciformis* through biometabolism, influenced by numerous factors, including global regulators, signaling molecules, and rate-limiting enzymes. While the complex, long-chain metabolic pathway of gibberellin biosynthesis in *Gibberella fuciformis* has been identified, the rate-limiting step remains uncertain in original strains with varying productivity, thus limiting the efficiency of gibberellin synthesis. Summary of the Invention

[0004] To address the problem of gibberellin GA in Fusarium oxysporum in existing technologies 4+7 To address the problem of low synthesis efficiency, this invention provides a gibberellin GA 4+7 The genetic engineering construction method and application of high-efficiency production strains have broken through the bottleneck of synergistic regulation of the rate-limiting step in the gibberellin metabolic pathway and eliminated the feedback inhibition of the key enzyme HmgR.

[0005] The technical solution adopted in this invention is: a gibberellin GA 4+7 A genetic engineering method for constructing a high-efficiency production strain, characterized in that the method comprises: using *Fujikura fusca* strain with the P450-3 gene knocked out as the substrate strain, overexpressing at least one gene selected from the following: thmgr (a truncated form of hydroxymethylglutaryl-CoA reductase), ggs2 (a geranyl diphosphate synthase), and cps / ks (a diterpenoid cyclase), to construct gibberellin GA. 4+7 Highly efficient production strains.

[0006] In previous work, by knocking out the p450-3 gene in *Fujikura scab*, a strain producing only GA was obtained. 4+7The production strain is △p450-3. This invention uses △p450-3 as the starting strain, enhances the expression of key genes in the metabolic pathway, explores the rate-limiting step in gibberellin synthesis, and attempts to further improve GA production. 4+7 Production, to obtain GA 4+7 High-yielding bacteria. Combined with... Figure 1 Gibberellin synthesis begins with the condensation of two acetyl-CoA molecules to form hydroxymethylglutaryl-CoA, which is then reduced to mevalonate by Hmgr. Since mevalonate is a key intermediate, this step is the rate-limiting step in the isoprene pathway. Mevalonate is converted to FDP via IPP, DMADP, and GDP catalyzed by farnesyl diphosphate synthases Fpps. FDP is used for the production of primary metabolites, catalyzing the synthesis of GGDP. *Fusarium oxysporum* has two distinct GGDP synthase encoding genes, ggs1 and ggs2, the latter being part of the GAs biosynthetic gene cluster. The absence of ggs2 results in *Fusarium oxysporum* ceasing gibberellin production, while the absence of ggs1 leads to cell death. GGDP synthesized by ggs2 is then converted to kauriene via a two-step cyclization reaction of p-copolydiphosphate catalyzed by diterpenoid cyclases CPS / KS. This process is further facilitated by cytochrome P450-4 and GAs. 14 Catalyzed by synthase P450-1, through kaurenoic acid and GA 12 GA is generated through a series of oxidation steps. 14 Subsequently, C20 is continuously oxidized by C20 oxidase P450-2 to form the first biologically active gibberellin GA4. In the final step, GA4 is catalyzed by desaturase DES to generate GA7, which is then converted to the final product GA3 by C13 hydroxylation by 13-hydroxylase P450-3. Therefore, this invention uses the Fujikura Gibberella Δp450-3 mutant strain as the substrate bacterium. Through single-gene overexpression experiments, four key rate-limiting genes in the gibberellin synthesis pathway—fpps, ggs2, cps / ks, and thmgr—were identified. A combination of rate-limiting genes with significant synergistic effects (thmgr, ggs2, cps / ks) was selected for co-expression to construct a gibberellin GA3 strain. 4+7 High-efficiency production strains

[0007] Preferably, the method includes: using *Fujikura gibberellinii* with the P450-3 gene knocked out as the substrate fungus, constructing a vector co-expressing the genes thmgr, ggs2, and cps / ks, and integrating the vector into the substrate fungus genome via protoplast transformation to construct gibberellin GA. 4+7 Highly efficient production strains.

[0008] Preferably, the vector further comprises a Fujikura gibberellin screening marker recovery system based on an inducible recombinase; the Fujikura gibberellin screening marker recovery system includes an inducible promoter, a site-specific recombinase regulated by the inducible promoter, a screening marker, and specific recombination sites located on both sides of the screening marker; the site-specific recombinase recognizes the specific recombination sites and cleaves the screening marker.

[0009] Preferably, the specific recombinase is an Flp recombinase or a Cre recombinase; the specific recombination site is an FRT or a loxP; the Flp recombinase recognizes the specific recombination site FRT; and the Cre recombinase recognizes the specific recombination site loxP.

[0010] Preferably, the method further includes: inducing the gibberellin GA 4+7 To efficiently produce strains with site-specific recombinases, gibberellin GA without selectable markers was obtained. 4+7 Highly efficient production strains.

[0011] Specifically, this invention uses the overexpression plasmid pUC-fFuCas-Amp-BleoR containing a bleomycin resistance tag as a backbone. The aforementioned key rate-limiting gene and the Fujikura gibberellin selection marker recovery system are constructed into this overexpression plasmid. It is then integrated into the *Bacillus subtilis* genome via protoplast transformation, and the strain is induced to express the aforementioned site-specific recombinase. The site-specific recombinase recognizes specific recombination sites located flanking the selection marker (i.e., the bleomycin resistance tag) and recovers the recombination, obtaining gibberellin GA without the selection marker. 4+7 Highly efficient production strains.

[0012] Preferably, the nucleotide sequence of the gene thmgr is shown in SEQ ID NO. 1, the nucleotide sequence of the gene ggs2 is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene cps / ks is shown in SEQ ID NO. 3.

[0013] The present invention also provides gibberellin GA constructed by the method. 4+7 Highly efficient production strains.

[0014] The present invention also provides the aforementioned gibberellin GA 4+7 Highly efficient production strains in the bio-fermentation preparation of gibberellin (GA) 4+7 Applications in [the study / conversion]. The applications include: [using] the gibberellin GA... 4+7 Highly efficient production strains were inoculated into fermentation medium and fermented to obtain gibberellin (GA). 4+7The fermentation medium comprises 110-130 g / L rice flour, 20-40 g / L corn starch, 8-12 g / L soybean meal, 1-3 g / L K₂SO₄, 0.5-1.5 g / L MgSO₄·7H₂O, and 0.004-0.006 g / L ZnSO₄·7H₂O. This invention also relates to GA... 4+7 Key components of the fermentation medium were screened, and an optimal ratio of corn starch, rice flour, and soybean meal was determined using a three-factor, three-level orthogonal design. The optimal ratio was then determined within the optimal GA range. 4+7 In the fermentation medium, the amounts of corn starch, rice flour, and soybean meal added, by weight, are 3%, 15%, and 1.35%, respectively; and the amounts of K2SO4, MgSO4·7H2O, and ZnSO4·7H2O added are 0.25%, 0.15%, and 0.0005%, respectively. In a preferred embodiment, the fermentation medium comprises 120 g / L rice flour, 30 g / L corn starch, 10 g / L soybean meal, 2 g / L K2SO4, 1 g / L MgSO4·7H2O, and 0.005 g / L ZnSO4·7H2O.

[0015] Preferably, the application includes adding alkali solution at 90-100 h of fermentation to maintain the pH of the fermentation medium at 6.0-6.5. Specifically, adding NaOH at 96 h of fermentation, and supplementing NaOH during the rapid growth phase of the cells (96 h) to maintain the pH in the range of 6.12-6.42, can relieve acid inhibition and promote yield accumulation.

[0016] The beneficial effects of this invention: This invention relieves feedback inhibition by truncating thmgr, and promotes precise carbon flux flow to GA under the co-expression of the triple gene (thmgr, ggs2, cps / ks). 4+7 The synthesis of GA was determined through orthogonal experiments. 4+7 The optimal ratio of key components in the fermentation medium—corn starch, rice flour, and soybean meal—was used to construct the final gibberellin GA. 4+7 High-efficiency production strains in optimized GA 4+7 GA in fermentation medium 4+7 The yield reached 3.6 g / L, while the starting strain in unoptimized fermentation medium yielded GA 4+7 The yield was only 0.92 g / L. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the biosynthetic pathway and rate-limiting steps of Gibberellin from Gibberellinus fusiforme in this invention.

[0018] Figure 2 This is a map of the ggs2-hmgr-cps / ks co-expression cassette constructed in Example 1 of this invention.

[0019] Figure 3 The image shows the recovery plasmid map of the OE:ggs2-hmgr-cps / ks co-expression tag constructed in Example 1 of this invention.

[0020] Figure 4 Example 2 of the present invention: Gibberellin GA 4+7 Yield of high-efficiency production strains. Detailed Implementation

[0021] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0022] Example 1: Construction of Gibberellin GA 4+7 High-efficiency production strains

[0023] 1. Constructing overexpression plasmids

[0024] The nucleotide sequences fpps, hmgr, ggs2, cps / ks, thmgr, and the bleomycin resistance Bleor sequence were amplified using upstream and downstream primers F and R in Table 1, respectively. The previously obtained expression cassette gene fragments and the bleomycin resistance gene were ligated to the linearized vector pUC-fFuCas-Amp-BleoR using one-step cloning to obtain expression plasmids OE:fpps, OE:hmgr, OE:ggs2, OE:cps / ks, and OE:thmgr. A rate-limiting gene combination with a significant synergistic effect (hmgr, ggs2, cps / ks) was selected for co-expression to construct a co-expression gene cassette. Figure 2 The expression plasmid OE was constructed by combining the inducible recombinase with the Fujikura gibberellin selection marker recovery system into the vector pUC-fFuCas-Amp-BleoR, resulting in the expression plasmid OE: thmgr-ggs2-cps / ks ( Figure 3 The Fujikura gibberellin screening and labeling recovery system for the inducible recombinase contains a Tet-on-guided tetracycline-inducible FLP recombinase expression cassette and a bleomycin expression cassette with FRT-specific recognition sites on both sides.

[0025] Table 1. Primer Table

[0026] 2. Preparation of Fusarium Fujikura protoplasts

[0027] Using a sterile toothpick, a thumb-sized patch of *Fujikura fusarium* Δp450-3 was scooped from PDA (PDA medium: 200 g / L potato dextrose, 20 g / L glucose, 15 g / L agar powder) medium and cultured in YEPD medium (YEPD medium: 30 g / L yeast extract, 10 g / L peptone, 20 g / L glucose) for 2 days. In a clean bench, the cultured YEPD medium was poured into a Buchner funnel lined with double-layered filter paper and filtered until dry. The cells were then washed three times with 0.8 mol / L sodium chloride solution and filtered until dry. Approximately 1 g of cells was scraped from the cells using a sterile pipette tip and added to 10 mL of cell wall enzymatic hydrolysis solution (prepared with 1% Driselase, 2% Yatalase, 1% Snailase, and 0.8 M NaCl solution). The cells were incubated at 30°C and 150 rpm for 2–3 hours, gently inverting the container every half hour to ensure thorough mixing. Filter the fully enzymatically digested bacterial culture through a double-layer Miracloth filter into sterile 50 mL centrifuge tubes to remove residual cells and insoluble matter. Add 10 mL of 0.8 mol / L sodium chloride solution, mix thoroughly with a pipette tip to fully suspend the protoplasts, centrifuge at 900 × g, 4 °C for 10 min, and discard the supernatant. Repeat this step twice. Add 10 mL of LTSC solution, mix thoroughly with a pipette tip to fully suspend the protoplasts, centrifuge at 900 × g, 4 °C for 10 min, and discard the supernatant. Repeat this step twice. Add 2 mL of LTSC solution, mix thoroughly with a pipette tip to fully suspend the protoplasts, and dilute to 10. 7 Quantity / mL, store at 4℃ for later use.

[0028] 3. Protoplast transformation

[0029] The expression plasmids OE:fpps, OE:hmgr, OE:ggs2, OE:cps / ks, OE:thmgr, and OE:thmgr-ggs2-cps / ks constructed in step 1 were introduced into the protoplasts of *Fujikura fusarium* Δp450-3 using PEG-mediated transformation. The transformation method is as follows.

[0030] Take 160 μL of protoplast resuspension, 100 μL of expression plasmid pOEorippt1, and 60 μL of 60% PEG6000 solution into a 2 mL centrifuge tube and mix well. The plasmid and donor fragment should each reach 10 μg. Positive control group: Take 160 μL of protoplast resuspension, 100 μL of LTC solution, and 60 μL of 60% PEG6000 solution into a 2 mL centrifuge tube and mix well. Negative control group: Take 160 μL of protoplast resuspension, 100 μL of LTC solution, and 60 μL of 60% PEG6000 solution into a 2 mL centrifuge tube and mix well. Place the 2 mL centrifuge tubes with the mixture on ice and invert them every 10 min to mix. Repeat 3 times. Add 1.5 mL of 60% PEG6000 solution, mix by pipetting, and let stand at room temperature for 25 min. Add 6 mL of soft agar MYG liquid medium containing 100 ng / μL hygromycin resistance (MYG: 5 g / L maltose, 5 g / L yeast extract, 10 g / L glucose, 171 g / L sucrose, 20 g / L agar, solvent: water) to a 50 mL centrifuge tube, along with 3 mL of preheated STC solution and the above transformation system. Shake to mix well and then pour the mixture onto a plate containing MYG hard agar medium of the same hygromycin concentration. Transfer the plate to a 28°C incubator and incubate upright for 3–5 days to obtain positive transformants.

[0031] 4. Rescreening of positive transformants

[0032] Ten positive transformants were picked with sterile toothpicks and seeded onto PDA agar plates, incubated at 28°C for 3 days. A portion of mycelium was picked with a sterile toothpick and placed into a 2 mL centrifuge tube containing 200 μL of lysis buffer, and the mycelium was thoroughly homogenized using a tissue homogenizer. The genome of the transformants was crudely extracted using the TSINGKET SP501-50 high-purity plasmid DNA mini-extraction kit, and the concentration was determined. The genomes of the positive transformants were validated by PCR using verification primers YZppt1F and YZppt1R, and then sequenced. Gibberellin GA was confirmed to be present. 4+7 The high-efficiency production strains are OE:fpps, OE:hmgr, OE:ggs2, OE:cps / ks, OE:thmgr, and OE:thmgr-ggs2-cps / ks.

[0033] 5. Recycle screening markers

[0034] Tetracycline-induced Tet-on expression of FLP recombinase recovery and screening markers: The strain from step 4 was inoculated into YEPD medium, and tetracycline was added to a final concentration of 1 μg / mL. The culture was incubated at 28℃ and 250 rpm for 48 h to induce the Tet-on system to express the recombinase FLP. The FRT sites flanking bleomycin resistance were recognized, and bleomycin resistance was recovered for the next gene editing. The cultured bacterial cells were prepared into protoplasts using the above method, appropriately diluted, and then revived on MYG medium.

[0035] Screening marker recovery and validation: Single colonies revived in the previous step were picked and placed on PDA solid medium and PDA solid medium supplemented with bleomycin resistance, respectively. After tetracycline-induced Tet-on expression of FLP recombinase and recovery of the screening marker, the engineered strain could not grow on bleomycin-resistant PDA solid medium. Colonies that could grow on non-resistant PDA solid medium but not on resistant PDA solid medium were selected for the next step of the experiment.

[0036] Example 2: Gibberellin (GA) 4+7 Fermentation of high-efficiency production strains

[0037] 1. GA 4+7 Fermentation medium optimization

[0038] Single-factor experiments were conducted on the addition amounts of various components of the fermentation medium, revealing that corn starch, rice flour, and soybean meal had significant effects on the fermentation medium. Therefore, gibberellin (GA) was used as the primary component. 4+7 Using output as the indicator, an orthogonal experiment with three factors and three levels was designed, as shown in Table 2.

[0039] Table 2. Factors and Levels

[0040] Factor Level Corn starch (A, %) Rice noodles (B, %) Soybean meal (C, %) 1 0 12 1 2 3 15 1.35 3 6 18 1.7

[0041] Orthogonal arrays were automatically generated using an orthogonal design tool, and the results are shown in Table 3. Range analysis revealed that the order of influence of each factor on gibberellin yield was corn starch > rice flour > soybean meal. The optimal experimental combination was A2B2C2. Under these conditions, the GA array constructed in the laboratory previously was used... 4+7 The high-yield strain OE:ppt1-SWEET101 was used for fermentation verification.

[0042] The optimal addition amounts of corn starch, rice flour, and soybean meal were determined to be 3%, 15%, and 1.35%, respectively. 4+7In the fermentation medium, the addition amounts of corn starch, rice flour, and soybean meal, by weight, were 3%, 15%, and 1.35%, respectively; the addition amounts of K₂SO₄, MgSO₄·7H₂O, and ZnSO₄·7H₂O were 0.25%, 0.15%, and 0.0005%, respectively. Under optimal culture conditions, GA 4+7 Production increased by approximately 15.4%, as shown in Table 3.

[0043] Table 3. Orthogonal experimental design and results

[0044] serial number Corn starch (A) Rice noodles (B) Soybean meal (C) GAs production (g / L) 1 1 1 1 0.719 2 1 2 2 0.97 3 1 3 3 0.86 4 2 1 2 1.22 5 2 2 3 1.16 6 2 3 1 1.25 7 3 1 3 0.779 8 3 2 1 0.973 9 3 3 2 0.894 k1 0.850 0.906 0.981 / k2 1.210 1.034 1.028 / k3 0.882 1.001 0.933 / R 0.360 0.128 0.095 /

[0045] 2. Fermentation by bacterial strain

[0046] The gibberellin GA constructed in Example 1 4+7 Five positive transformants of the high-efficiency production strains OE:fpps, OE:hmgr, OE:ggs2, OE:cps / ks, OE:thmgr, and OE:thmgr-ggs2-cps / ks were inoculated into seed culture medium (seed culture medium: corn starch 20 g / L, sucrose 15 g / L, peanut powder 15 g / L, soybean meal 3 g / L, KH2PO4 1 g / L, MgSO4 1 g / L) and cultured at 28℃ and 250 rpm for 48 hours. Then, they were transferred to the optimal GA strain at a 6% (v / v) inoculation rate. 4+7 Fermentation was carried out at 28℃ and 250 rpm for 7 days in the fermentation medium. At the 96th hour of fermentation, 100 μL of 7 mol / L NaOH was added to every 60 mL of fermentation medium to maintain the pH between 6.12 and 6.42. After fermentation, the supernatant was centrifuged, and the contents of GA3, GA4, and GA7 were determined by high-performance liquid chromatography (HPLC, Agilent 1260). The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase for GA3 detection was methanol:0.05% phosphoric acid water = 40:60. 4+7 The mobile phase used for detection was methanol:water:phosphoric acid = 68:32:0.05. Results are shown below. Figure 4 It can be seen that, compared to the full-length hmgr gene, overexpression of the truncated thmgr gene is actually beneficial to GA. 4+7 The yield was increased, and compared with single gene overexpression, the co-expression of the rate-limiting gene combination (thmgr, ggs2, cps / ks) produced a significant synergistic effect, ultimately leading to increased GA yield. 4+7 The yield reached 3.6 g / L.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A type of gibberellin (GA) 4+7 A genetic engineering method for constructing highly efficient production strains, characterized in that, The method includes: using *Fujikura fuciformis* strain with the P450-3 gene knocked out as the substrate fungus, and overexpressing the truncated hydroxymethylglutaryl-CoA reductase gene. thmgr Geranyl diphosphate synthase gene ggs2 diterpenoid cyclase gene cps / ks At least one of the genes in the gene was used to construct gibberellin GA. 4+7 Highly efficient production strains.

2. The method as described in claim 1, characterized in that, The method includes: using *Fusarium oxysporum* strain with the P450-3 gene knocked out as the substrate fungus to construct a co-expression gene. thmgr ,Gene ggs2 ,Gene cps / ks The vector was constructed and integrated into the genome of *Bacillus subtilis* using protoplast transformation to obtain gibberellin GA. 4+7 Highly efficient production strains.

3. The method as described in claim 2, characterized in that, The vector also has a Fujikura gibberellic acid selection marker recovery system based on an inducible recombinase; the Fujikura gibberellic acid selection marker recovery system includes an inducible promoter, a site-specific recombinase regulated by the inducible promoter, a selection marker, and specific recombination sites located on both sides of the selection marker; the site-specific recombinase recognizes the specific recombination sites and cleaves the selection marker.

4. The method as described in claim 3, characterized in that, The specific recombinase is either Flp recombinase or Cre recombinase; the specific recombination site is either FRT or loxP; the Flp recombinase recognizes the specific recombination site FRT; the Cre recombinase recognizes the specific recombination site loxP.

5. The method as described in claim 3, characterized in that, The method further includes: inducing the gibberellin GA 4+7 To efficiently produce strains with site-specific recombinases, gibberellin GA without selectable markers was obtained. 4+7 Highly efficient production strains.

6. The method as described in claim 1, characterized in that, The gene thmgr The nucleotide sequence is shown in SEQ ID NO.1, gene. ggs2 The nucleotide sequence is shown in SEQ ID NO. 2, gene. cps / ks The nucleotide sequence is shown in SEQ ID NO.

3.

7. Gibberellin GA constructed by the method according to any one of claims 1 to 6 4+7 Highly efficient production strains.

8. Gibberellin GA constructed by the method according to any one of claims 1 to 6 4+7 Highly efficient production strain or the gibberellin GA described in claim 6 4+7 Highly efficient production strains in the bio-fermentation preparation of gibberellin (GA) 4+7 Applications in [the context of the text].

9. The application as described in claim 8, characterized in that, The application includes: using the gibberellin GA 4+7 Highly efficient production strains were inoculated into fermentation medium and fermented to obtain gibberellin (GA). 4+7 The fermentation medium comprises 110-130 g / L rice flour, 20-40 g / L corn starch, 8-12 g / L soybean meal, 1-3 g / L K2SO4, 0.5-1.5 g / L MgSO4·7H2O, and 0.004-0.006 g / L ZnSO4·7H2O.

10. The application as described in claim 8, characterized in that, The application includes adding alkali solution at 90-100 h of fermentation to maintain the pH of the fermentation medium at 6.0-6.5.

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