Gibberellin GA4+7 producing strain as well as construction method and application thereof

Abstract

The invention provides a gibberellin GA4 + 7 producing strain as well as a construction method and application thereof. Through a plurality of genetic engineering strategies, the method comprises the following steps: (1) heterologous expression of a plant source gibberellin transporter; (2) overexpressing an endogenous post-translational modification enzyme ppt1 to indirectly trigger mRNA degradation of a downstream key enzyme gene P450-3; and a plurality of strategies are synergistically combined, so that the industrial gibberella zeylanica mainly producing GA3 is successfully transformed into a specific engineering strain with high yield of GA4 + 7, and the generation of GA3 is completely eliminated. In combination with fermentation process optimization, the maximum yield of GA4 + 7 of the engineering strain on a shake flask level can reach 3.53 g / L, which is improved by 130 times or more compared with that of an original strain, and the engineering strain has excellent genetic stability and huge industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering, specifically to the production of gibberellins through microbial fermentation, and more specifically to gibberellin GA4 and GA7 mixtures that can specifically and efficiently produce high yields. 4+7 Production strains, their construction methods, and applications. Background Technology

[0002] Gibberellin (GA) s Gibberellins are a class of key endogenous hormones that regulate plant growth and development, and they have significant commercial value as plant growth regulators in agriculture, horticulture, and forestry. Industrially, gibberellins are mainly produced through liquid submerged fermentation using the filamentous fungus *Fusarium fujikuroi*.

[0003] Currently, the main commercially available gibberellin products are GA3 and GA2. 4+7 (A mixture of GA4 and GA7), with GA3 as the dominant product. Industrial strains primarily produce gibberellin GA3, whose production process is mature and relatively inexpensive. However, GA... 4+7 Because it exhibits superior biological activity compared to GA3 in promoting fruit setting, reducing fruit rust, regulating flowering, and overcoming dwarfing, its market demand is growing rapidly.

[0004] However, naturally occurring or conventionally induced *Fujikura fusarium* primarily synthesizes and accumulates GA3, whose GA... 4+7 Production is extremely low; currently reported GA 4+7 The yield is only 600-800 mg / L. GA 4+7 Due to its low production efficiency, its market price is much higher than that of GA3, and the development of high-yield strains and supporting processes has significant economic value.

[0005] The gibberellin biosynthesis pathway in *Fusarium oxysporum* is quite complex. At the end of this pathway, GA4 is a precursor to GA7 and GA1, and GA7 is a precursor to GA3, catalyzed by P450-3 monooxygenase. Therefore, the metabolic flux favors the synthesis of the final product GA3, resulting in very limited accumulation of intermediate products GA4 and GA7.

[0006] In the prior art, to improve GA 4+7The primary strategy for increasing yield is gene knockout, such as knocking out the P450-3 gene that catalyzes the conversion of GA7 to GA3, or knocking out the des gene that catalyzes the conversion of GA4 to GA7. However, while these simple knockout strategies can block some metabolic pathways, the yield increase is limited and may lead to metabolic imbalance, insufficient accumulation of precursor substances, or their diversion to other competing secondary metabolic pathways (such as fumonisin, fusarium oxysine, and bicardiin), which still cannot meet the needs of industrial production. Therefore, there is an urgent need in this field for more efficient metabolic engineering strategies to completely reconstruct the metabolic network of strains and achieve GA3 production. 4+7 Highly efficient and specialized production. Summary of the Invention

[0007] The purpose of this invention is to overcome the limitations of existing technologies in the production of GA by Fujikura Fusarium. 4+7 Addressing the issues of low yield and limited, inefficient metabolic flux regulation, this study offers a series of novel genetic engineering strategies to construct structures capable of specifically and efficiently synthesizing GA. 4+7 The recombinant strain, combined with fermentation process optimization, achieved GA 4+7 Industrialized production level.

[0008] The technical solution adopted in this invention is: a gibberellin GA 4+7 The method for constructing the production strain, using *Fusarium fujikuroi* as the starting strain, includes at least one of the following steps (1)-(2):

[0009] (1) Overexpression of the Sfp-type 4'-phospho-pantothenic acid thioethylamine transferase encoding gene ppt1;

[0010] (2) Overexpression of the gene encoding the plant-derived gibberellin transporter;

[0011] The gibberellin GA is thus obtained. 4+7 Production strain;

[0012] The gibberellin transporter is selected from at least one of the SWEET protein family or the NPF protein family.

[0013] The GA provided by this invention 4+7 The producing strain has been genetically engineered to heterologously express one or more plant-derived (preferably Arabidopsis thaliana) gibberellin transporters selected from the SWEET or NPF protein families. This strategy facilitates the active efflux of intracellular gibberellin transporters. 4+7 They are transported extracellularly, thereby reducing the chance of them being catalyzed by downstream enzymes to generate GA3, thus realizing the conversion from GA3 to GA. 4+7The invention also upregulated the expression level of the endogenous Sfp-type 4'-phosphopantoylthioethylamine transferase gene ppt1. Experiments showed that overexpression of ppt1 in *Fusarium oxysporum* triggered specific and efficient degradation (degradation rate exceeding 99.5%) of the P450-3 gene mRNA. This is an unprecedented "post-translational modification-mediated targeted mRNA degradation" mechanism. Using this mechanism, the invention achieved complete blockade of the GA3 synthesis pathway. In a preferred embodiment, the invention integrated two or more of the above-mentioned modification strategies; for example, overexpression of the ppt1 gene in strains heterologously expressing the SWEET1 transporter protein showed that this strategy had a significant synergistic effect.

[0014] Preferably, the gibberellin transporter is derived from Arabidopsis thaliana.

[0015] Preferably, the nucleic acid sequence of the ppt1 gene is shown in SEQ ID NO.1.

[0016] Preferably, the SWEET protein family includes SWEET1, SWEET5, SWEET6, SWEET13, and SWEET14, with SWEET1 being the most preferred. The nucleic acid sequence of SWEET1 is preferably shown in SEQ ID NO.2.

[0017] Preferably, the NPF protein family includes NPF1.2, NPF4.1, NPF5.5, NPF5.16, NPF6.2, NPF6.4, and NPF8.1, with NPF8.1 being the most preferred. The nucleic acid sequence of NPF8.1 is preferably as shown in SEQ ID NO.3.

[0018] Preferably, the method for overexpressing the Sfp-type 4'-phosphopantoylthioethylamine transferase encoding gene ppt1 includes: constructing a ppt1 overexpression element under the regulation of a strong promoter, and transforming it into a starting strain. Preferably, the ppt1 gene is placed under the regulation of the strong promoter gpdA, a ppt1 overexpression plasmid is constructed, and the ppt1 overexpression plasmid is introduced into the starting strain through methods such as protoplast transformation.

[0019] Preferably, the method for overexpressing the gibberellin transporter encoding gene from a plant source includes: constructing a gibberellin transporter overexpression element under the regulation of a strong promoter, and transforming it into a starting strain. More preferably, the gibberellin transporter encoding gene is placed under the regulation of the strong promoter gpdA, a gibberellin transporter overexpression plasmid is constructed, and the plasmid is introduced into the starting strain via protoplast transformation or other methods.

[0020] Preferably, the method includes: using *Fusarium fujikuroi* as the starting strain, overexpressing the ppt1 and SWEET1 encoding genes to obtain the gibberellin GA. 4+7 Production strains.

[0021] The present invention also provides gibberellin GA constructed by the method. 4+7 Production strains.

[0022] The present invention also provides the aforementioned gibberellin GA 4+7 The producing strain is used in the fermentation production of gibberellin (GA). 4+7 Applications include: the gibberellin GA 4+7 The production strain was inoculated into the fermentation medium and fermented to obtain gibberellin GA. 4+7 .

[0023] Preferably, an alkaline regulator is added during the 96-144 h fermentation period. In the later stages of fermentation (e.g., 96 h or 144 h), the addition of an alkaline regulator neutralizes acidic byproducts and maintains a favorable pH environment. The alkaline regulator includes NaOH and NH3·H2O; NaOH is preferred as it has better regulatory effects and can maintain the pH between 6 and 7 within 96-168 h of fermentation.

[0024] As a preferred method, a baffled shaker (such as a three-convex baffle shaker) is preferred as the fermentation container during the fermentation process to increase the dissolved oxygen content.

[0025] Preferably, 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. The fermentation medium may also include a surfactant, such as 0.4%-0.6% stearic acid.

[0026] The beneficial effects of this invention are:

[0027] 1. Through the modification strategies proposed in this invention (overexpression of ppt1 and / or heterologous overexpression of SWEET1), the strain can be completely transformed from a GA3-producing strain to one that does not produce GA3 and exclusively produces GA. 4+7 The strain solved the problem of product impurity, achieving complete reprogramming and specific production of metabolites. Compared to the starting strain GA... 4+7 (Yield of 0.026 g / L), through a single strategy modification, GA 4+7Yield increased more than 30 times; through a collaborative engineering strategy, yield reached 1.12 g / L. Ultimately, combined with fermentation process optimization, GA... 4+7 The final yield reached 3.29 g / L. Under optimized dissolved oxygen conditions, even with a baffle shake flask, the yield could reach 3.53 g / L, which is more than 130 times higher than the original strain. This is the highest level of shake flask fermentation known to date, achieving a significant increase in yield.

[0028] 2. This invention is the first to discover and utilize a novel regulatory mechanism that ppt1 protein overexpression can induce specific degradation of P450-3 mRNA, providing a powerful regulatory tool for fungal metabolic engineering that surpasses traditional gene knockout or CRISPRi, revealing and applying a completely new biological regulatory mechanism.

[0029] 3. The gibberellin GA obtained in this invention 4+7 The production strain, through stress selection and subculturing, demonstrated good genetic stability and excellent production performance, making it a high-value-added product, GA. 4+7 It has laid a solid foundation for industrialized production and has enormous potential for industrial applications. Attached Figure Description

[0030] Figure 1 This is a heatmap showing the GA3 yield of each transformant after heterologous expression of 18 different plant transport proteins in Example 1 of the present invention.

[0031] Figure 2 In Example 1 of this invention, after heterologous expression of 18 different plant transport proteins, the GA transformants were... 4+7 Heat map of production output.

[0032] Figure 3 This is a bar graph comparing the gibberellin yield of transformant SWEET1-01 and the starting strain in Example 1 of the present invention.

[0033] Figure 4 This is a trophic stability diagram of the high-yield transformant SWEET1-01 in Example 1 of the present invention after 10 generations of passage culture.

[0034] Figure 5 The relative expression levels of transporter genes and their GA in the high-yield transformants SWEET1-01 and NPF8.1-02 of Example 1 of this invention are shown. 4+7 A graph showing the relationship between production output and output.

[0035] Figure 6 This is a comparison chart of gibberellin fermentation yield of strains overexpressing the ppt1 gene in Example 2 of the present invention.

[0036] Figure 7 The yields of each transformant in Example 3 of this invention, which combines overexpression of transporter protein and PPTase.

[0037] Figure 8 This is a graph showing the effect of adding different alkaline substances at different time points on the pH value in Example 4 of the present invention.

[0038] Figure 9 This is a graph showing the change in gibberellin production after integrating and optimizing the strategy in Embodiment 4 of the present invention. Detailed Implementation

[0039] 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, well-known to those skilled in the art, and the reagents and materials used are commercially available.

[0040] All starting strains used in the examples were wild-type Fusarium fujikuroi Ff1101, derived from Qianjiang Biochemical Co., Ltd., with the starting strain being GA. 4+7 The yield was 0.026 g / L.

[0041] Components of each culture medium:

[0042] a) YEPD liquid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L;

[0043] b) PDA solid culture medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L;

[0044] c) Seed culture medium: sucrose 40 g / L, soybean meal 12 g / L, KH2PO4 1.3 g / L, MgSO4 1.5 g / L;

[0045] d) Fermentation medium: rice flour 120 g / L, corn starch 30 g / L, soybean meal 10 g / L, K2SO4 2 g / L, MgSO4·7H2O 1 g / L, ZnSO4·7H2O 0.005 g / L.

[0046] Molecular manipulation methods: Gene cloning, plasmid construction, PCR, etc., all employed standard molecular biology methods. Recombinant plasmids were constructed using the Vazyme ClonExpress Ultra One Step Cloning Kit C115.

[0047] Transformation method: Protoplast-polyethylene glycol (PEG) mediated transformation was employed. After mycelia were cultured in YEPD for 48 hours, they were enzymatically digested with 0.8M NaCl solution containing 200 mg of a mixed enzyme (50 mg snailase, 100 mg lysozyme, and 50 mg lysozyme) at 30°C and 120 rpm for 4 hours to prepare protoplasts. Transformed strains were then screened on MYG regeneration plates containing the appropriate antibiotics.

[0048] Shake-flask fermentation method: Positive transformants were inoculated into seed culture medium and cultured at 28℃ and 250 rpm for 48 hours. Then, they were transferred to fermentation medium at a 6% (v / v) inoculation rate and cultured at 28℃ and 250 rpm for 7 days. 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.

[0049] Example 1: Screening for high-yield GA by heterologous expression of plant-derived transport proteins 4+7 strain

[0050] This embodiment aims to identify the most effective transporter for GA by screening various plant-derived transport proteins. 4+7 Candidate proteins for production switching and yield increase.

[0051] 1. Plasmid Construction: Total RNA was extracted from Arabidopsis tissues and cDNA libraries were obtained by reverse transcription using the Novizan R223-01 kit. Using the cDNA as templates, the encoding genes of 18 transport proteins were amplified by PCR, including 10 NPF family proteins (NPF1.2, NPF2.5, NPF3.1, NPF4.1, NPF5.5, NPF5.16, NPF6.2, NPF6.4, NPF7.2, NPF8.1) and 8 SWEET family proteins (SWEET1, SWEET5, SWEET6, SWEET9, SWEET10, SWEET11, SWEET13, SWEET14). Each gene fragment was ligated into the pUC-fFuCas9-HTBNLS-hph plasmid driven by the strong promoter gpdA (SEQ ID NO. 4) to construct a series of expression plasmids.

[0052] 2. Transformation and Screening of Strains: The 18 constructed expression plasmids were transformed into protoplasts of *Fujikura fusiforme* Ff1101. Screening was performed on MYG regeneration plates containing hygromycin (100 μg / mL). For each transport protein, at least 5 positive transformants were randomly selected, resulting in a total of 90 transformants.

[0053] 3. Fermentation and Yield Determination: Ninety transformants were subjected to shake-flask fermentation. The results are as follows: Figure 1 and Figure 2 As shown, the GA3 yield decreased in most transformants, while GA 4+7 Yields increased to varying degrees. Among them, 12 high-yielding strains (NPF1.2, NPF4.1, NPF5.5, NPF5.16, NPF6.2, NPF6.4, NPF7.2, NPF8.1, SWEET1, SWEET5, SWEET6, SWEET13, and SWEET14) were screened, and their GA... 4+7 The yields were all more than 7 times that of the original strain.

[0054] 4. Optimal strain analysis: The strain with the best performance was SWEET1-01. For example... Figure 3 As shown, the yield of GA3 in the fermentation broth of this strain decreased to 0, the yield of GA4 was 0.0765 g / L, the yield of GA7 was 0.7558 g / L, and the yield of GA... 4+7 The total yield reached 0.8323 g / L, which was 2.43 times, 252.3 times, and 31.3 times that of the original strain, respectively.

[0055] 5. Stability and Mechanism Verification: Strain SWEET1-01 was subjected to 10 consecutive generations of stress subculturing containing hygromycin B, and its GA... 4+7 Production remained stable. Figure 4 This demonstrates good genetic stability. qPCR analysis showed that the mRNA expression level of the transporter protein in high-yielding strains (such as SWEET1-01) was significantly higher than that in low-yielding strains (approximately 8-fold higher), proving a positive correlation between yield and transporter protein expression level. Figure 5 Intracellular gibberellin detection revealed that wild-type strains accumulated large amounts of GA intracellularly. 4+7 The significantly reduced intracellular content of the transformant strain directly demonstrates the efflux function of the transporter protein.

[0056] Example 2: Metabolic reprogramming achieved by overexpressing the endogenous ppt1 gene

[0057] This embodiment verifies the technical solution of blocking GA3 synthesis by overexpressing ppt1.

[0058] 1. Strain Construction: Using the *Fujikura fusarium* genome as a template, the ppt1 gene shown in SEQ ID NO.1 was amplified by PCR. This gene was ligated into a plasmid vector regulated by the strong promoter gpdA to construct a ppt1 overexpression plasmid. Transformation and screening were performed using the same method as in Example 1.

[0059] 2. Fermentation and Yield Determination: Positive transformants were subjected to shake-flask fermentation and HPLC analysis. Results are as follows: Figure 6 As shown, in transformants overexpressing ppt1, GA3 production decreased to 0, while GA4 and GA7 production increased significantly. The optimal strain OE:ppt1-3 achieved GA4 production of 0.147 g / L and GA7 production of 0.788 g / L. 4+7 The total yield was 0.935 g / L, which was 6.89 times, 112.5 times, and 36 times that of the original strain, respectively.

[0060] 3. Mechanism Verification: RNA-seq analysis of the OE:ppt1-3 strain showed that the mRNA level of the P450-3 gene was extremely suppressed at all fermentation time points, with a reduction of 99.5%-99.9%. To verify that this was post-transcriptional degradation rather than transcriptional repression, the P450-3 gene was expressed again in the ppt1 overexpressing strain using an exogenous strong promoter. qPCR results showed that its mRNA level remained close to zero. This confirms that ppt1 overexpression mediates a highly efficient and specific P450-3 mRNA degradation mechanism.

[0061] Example 3: Achieving yield superposition through synergistic expression of SWEET1 and overexpression of ppt1

[0062] This embodiment aims to verify the synergistic effect of the two optimal strategies.

[0063] 1. Strains Construction: The hygromycin resistance marker (hph) of the ppt1 overexpression plasmid in Example 2 was replaced with the bleomycin resistance marker (bleoR). The optimal strain SWEET1-01 obtained in Example 1 was transformed with this new plasmid, and positive transformants expressing both elements were screened on a plate containing hygromycin and bleomycin.

[0064] 2. Fermentation and Yield Determination: All 20 obtained positive transformants were subjected to shake-flask fermentation and HPLC analysis. Results are as follows: Figure 8 As shown, the GA3 yield was 0 in all co-expression transformants. 4+7 The total yield remained stable between 1.05 g / L and 1.12 g / L. The highest yield was achieved by strain OEppt1S1-1 (…). Figure 7 GA of P01S1-1 4+7The yield reached 1.12 g / L, which is 34.5% higher than that of the SWEET1-01 strain and 43.08 times that of the wild-type strain.

[0065] Example 4: Fermentation process optimization

[0066] This embodiment optimizes the fermentation process of the optimal strain OEppt1S1-1 obtained in Example 3.

[0067] 1. pH Adjustment: Different concentrations of alkaline substances (NaOH, NH3·H2O) were added at different time points during fermentation. The pH results of the fermentation system are shown below. Figure 8 As shown in the figure, the results indicate that adding NaOH in the middle and late stages of fermentation (96 h and 144 h) has the most significant effect on increasing yield. The yield reached 1.63 g / L when NaOH was added at 96 h, which may be more conducive to maintaining the optimal pH environment for GA4 synthesis-related enzymes, producing an unexpected technical effect.

[0068] 2. Integration and Optimization: The above optimization strategies were combined, namely, adding a final concentration of 0.5% stearic acid to the fermentation medium, and adding 100 µL of 7 mol / L NaOH to every 60 mL of fermentation medium at the 96th hour of fermentation. Ultimately, GA... 4+7 The highest yield reached 3.29 g / L, an increase of 91% compared to the control group. Figure 9 Under another optimized condition, using a three-convex baffle shake flask to increase dissolved oxygen yielded a final output of 3.53 g / L.

[0069] 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 The method for constructing production strains is characterized by, Fumigranobacter fusiforme Fusarium fujikuroi For the starting strain, at least one of the following steps (1)-(2) is included: (1) Overexpression of the gene encoding Sfp-type 4'-phospho-pantothenicotinamide aminotransferase ppt1 ; (2) Overexpression of the gene encoding the plant-derived gibberellin transporter; The gibberellin GA is thus obtained. 4+7 Production strain; The gibberellin transporter is selected from at least one of the SWEET protein family or the NPF protein family.

2. The method as described in claim 1, characterized in that, The gibberellin transporter protein is derived from Arabidopsis thaliana. Arabidopsis thaliana.

3. The method as described in claim 1, characterized in that, The SWEET protein family includes SWEET1, SWEET5, SWEET6, SWEET13, and SWEET14; The NPF protein family includes NPF1.2, NPF4.1, NPF5.5, NPF5.16, NPF6.2, NPF6.4, NPF7.2, and NPF8.

1.

4. The method as described in claim 1, characterized in that, The gene encoding the overexpressed Sfp-type 4'-phosphopantoylthioethylamine transferase ppt1 The methods include: inserting genes ppt1 Constructed under the control of a strong promoter ppt1 Overexpress the element and transform it into the starting strain; The method for overexpressing the gibberellin transporter encoding gene from plants includes: constructing a gibberellin transporter overexpression element by placing the gibberellin transporter encoding gene under the regulation of a strong promoter, and transforming it into the starting strain.

5. The method as described in claim 1, characterized in that, Fusarium oxysporum Fusarium fujikuroi For the starting strain, overexpressed gene ppt1 And the SWEET1 encoding gene, thus obtaining the aforementioned gibberellin GA 4+7 Production strains.

6. Gibberellin GA constructed by the method described in any one of claims 1-5 4+7 Production strains.

7. Gibberellin GA constructed by the method described in any one of claims 1-5 4+7 The producing strain or the gibberellin GA as described in claim 6 4+7 The producing strain is used in the fermentation production of gibberellin (GA). 4+7 The application of [the technology] is characterized by, include: The gibberellin GA 4+7 The production strain was inoculated into the fermentation medium and fermented to obtain gibberellin GA. 4+7 .

8. The application as described in claim 7, characterized in that, An alkaline regulator was added during the 96-144 h fermentation period; the alkaline regulator included NaOH and NH3·H2O.

9. The application as described in claim 7, characterized in that, During the fermentation process, a baffle-shaking flask is used as the fermentation container.

10. The application as described in claim 7, characterized in that, 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.

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