Method for producing gibberellin GA4+7 mixture with different proportions by mixed bacteria fermentation and application thereof

Abstract

This invention discloses a method for producing a mixture of gibberellin GA4 and GA7 in different proportions through mixed bacterial fermentation. The method involves two high-yield gibberellin strains: one producing gibberellin GA4 (classified as *Fusarium fujikuroi* NRF01, deposited on May 19, 2022 at the China Center for Type Culture Collection, accession number CCTCC NO. M2022683) and the other producing gibberellin GA7 (classified as *Fusarium fujikuroi* NRF02). The sample was deposited at the China Center for Type Culture Collection on August 1, 2022, with accession number CCTCCNO.M20221211. By mixing high-yield strains of gibberellin GA4 and GA7 in different proportions and controlling fermentation conditions in stages, different proportions of gibberellin GA4 and GA7 mixtures can be obtained after 120 hours of fermentation. When using a two-stage culture to control fermentation conditions, the mixed strain can produce different proportions of gibberellin GA4 and GA7 mixtures in a shorter fermentation cycle. After 120 hours of fermentation, the gibberellin GA4+7 mixture has a potency of about 1600 mg / L.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for producing a mixture of gibberellin GA4+7 in different proportions by fermentation of mixed bacteria and its application. Background Technology

[0002] This invention relates to gibberellins GA4 and GA7, whose structural formulas are as follows:

[0003]

[0004] Gibberellins (GAs) are a class of plant hormones belonging to the diterpenoid class, widely found in higher plants, fungi, and bacteria. There are numerous types; currently, 136 species of gibberellins have been identified, collectively known as gibberellins (GAs). Gibberellins act as plant growth regulators with various regulatory functions. The types of gibberellins produced vary under different light and temperature conditions, at different stages of plant growth, and even in different parts of the same plant. GA7 is mainly produced in the shoot tip and immature seeds, while GA4 has been found in the roots, stems, leaves, and seeds. In terms of promoting stem growth, the order of gibberellin activity is GA3 > GA7 > GA4. GA3, due to its high activity, promotes excessive hypocotyl growth when breaking plant dormancy, reducing the plant's resistance to lodging. It also promotes rapid growth of epidermal cells, resulting in a thinner cuticle and making fruits prone to blemishes and cracking. GA7 strongly inhibits flower bud formation, while GA4 not only does not inhibit flower bud formation but actually promotes it. Therefore, the GA4+7 mixture can play a complementary role, and its high activity and unique advantages are attracting more and more attention.

[0005] Gibberellin GA4+7 belongs to the gibberellin series and is a mixture of GA4 and GA7. It is mainly used in apple shaping and is one of the main components of apple shaping agents. The application of gibberellin GA4+7 is an important technical measure for farmers to produce high-quality apples. In addition, when GA4+7 is combined with different plant growth regulators and sprayed on crops such as pears, sweet cherries, strawberries, bananas, and greenhouse fruits and vegetables, it also has a significant effect on improving quality and increasing yield. Different ratios of gibberellin GA4 and GA7 mixtures produce different effects. GA4+7, like GA3, is one of the most valuable and commercially viable products. Currently, GA4+7 products are widely used in China and some European and American countries.

[0006] Currently, gibberellin GA4+7 is mainly obtained through submerged liquid fermentation, but the yield is low, with fermentation levels around 600 mg / L, resulting in high production costs and severely hindering its large-scale commercial application. This study aims to further increase the yield of gibberellin GA4+7 and reduce its production costs through reasoned selection, targeted metabolic engineering, and fermentation process scale-up based on multi-scale parameter analysis, thereby promoting its large-scale application. This provides a new perspective and approach to fundamentally solve the technical challenges of microbial production of gibberellin GA4+7, enhancing our understanding of microbial cell metabolic regulation, achieving significant breakthroughs in microbial production capabilities, promoting the development of industrial biotechnology, and generating significant economic and social benefits. Domestic and international research on gibberellins and gibberellin products mainly focuses on gibberellin GA3, as it is currently the most widely used gibberellin product, with limited research on other gibberellins. However, the high activity and unique advantages of GA4+7 are attracting increasing attention, but its relatively low yield and high price remain major obstacles to its large-scale application. Currently, GA4+7 production capacity is low, while its application scope is expanding, and market demand will gradually increase. The price of GA4+7 is about five times higher than GA3, severely hindering its widespread application. The main reasons are low yield, low extraction rate, and instability, with significant fluctuations in product content. Research on GA4+7 is scarce both domestically and internationally, with only a few research papers and patents. For example, Hu Yonghong et al. from Nanjing University of Technology (CN 201210470269.7) proposed a method for producing gibberellin GA4+7 using Gibberella fuciformis and fermentation. This method improves the potency of GA4+7 by adding a mixed carbon source to the fermentation medium, adding vegetable oil and characteristic precursors (glycine and mevalonic acid) at specific times, and increasing oxygenation. After fermentation, GA4+7 accounted for about 70%, with a GA4:GA7 ratio of approximately 3:1. Foreign research on gibberellin mainly focuses on the mechanism of gibberellin GA3 synthesis. Tudzynski et al. obtained a 13-hydroxylase mutant strain Gibberella fujikuroi 6314 through ultraviolet mutagenesis. After 10 days of fermentation, the yield of GA7 reached 790 mg / L, GA4 34 mg / L, and GA3 35 mg / L. The fermentation period was also as long as 10 days (Tudzynski B, Mihlan M, et al., Characterization of the final two genes of the gibberellinbiosynthesis gene cluster of Gibberella fujikuroi. The Journal of Biological Chemistry. 2003, 278(31):28635-28643.).These methods can only produce a specific ratio of gibberellin GA4+7 mixture, and cannot obtain a mixture of gibberellin GA4 and GA7 in different ratios as needed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for producing a mixture of gibberellin GA4+7 in different proportions by fermentation of two strains. By mixing two strains in different proportions, the fermentation broth does not produce a mixture of gibberellin GA4 and GA7 in different proportions. The yield of gibberellin GA4+7 is increased by controlling the fermentation conditions in two stages.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for producing a mixture of gibberellin GA4+7 in different proportions by mixed bacterial fermentation, involving two high-yield gibberellin strains: one GA4-producing strain, classified as *Fusarium fujikuroi* NRF01, deposited at the China Center for Type Culture Collection (CCTCC) on May 19, 2022, with accession number CCTCC NO. M2022683; and one GA7-producing strain, classified as *Fusarium fujikuroi* NRF02, deposited at the CCTC on August 1, 2022, with accession number CCTCC NO. M20221211. The high-yield strains of gibberellin GA4 and GA7 in different proportions are mixed and cultured, and the fermentation conditions are controlled in stages. After fermentation for 120 hours, mixtures of gibberellin GA4 and GA7 in different proportions can be obtained.

[0009] Further steps include the following:

[0010] (1) Using an inoculation knife, scrape approximately 1 cm long and wide *Fusarium fujikuroi* NRF01 or *Fusarium fujikuroi* NRF02 mycelial blocks from the slant culture and inoculate them into shake flasks containing seed culture medium. The seed culture medium consists of: glucose 2.0-5.0%, soybean meal 1.0-3.0%, peanut meal 0.8-1.5%, dextrin 2.0-4.0%, KH2PO4 0.08-0.16%, (NH4)2SO4 0.01-0.03%, and MgSO4·7H2O 0.05-0.15%. The temperature is 28-30℃, the shaking speed is 300-400 rpm, and the culture time is 24-48 hours to obtain *Fusarium fujikuroi* NRF01 and *Fusarium* NRF02 mycelial blocks, respectively. 50-150 mL of NRF02-activated seed solution;

[0011] (2) The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed in different proportions, and the volume after mixing was 150 mL.

[0012] (3) Inoculate 150 mL of the mixed and activated seed solution into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consists of: glucose 2.0-4.0%, soybean meal 1.5-2.5%, peanut meal 1.0-2.0%, dextrin 2.0-4.0%, KH2PO4 0.10-0.20%, (NH4)2SO4 0.02-0.04%, MgSO4·7H2O 0.05-0.15%. The temperature is 28-30℃, the stirring speed is 250-350 rpm, and the aeration rate is 1.5-3.0 m³ / h. 3 Culture for 18-36 hours per hour to obtain a mixed seed solution;

[0013] (4) Inoculate the mixed seed culture at 8-12% (V / V) into a 100L fermenter (70L / 100L) containing fermentation medium. The composition of the fermentation medium is: carbon source 5.0%-10.0%, nitrogen source 2.5-4.0%, soybean oil 0.08-0.16%, MgSO4·7H2O 0.08-0.16%, (NH4)2SO4 0.02-0.05%, KH2PO4 0.15-0.40%, ZnSO4 0.005-0.01%.

[0014] Furthermore, the two-stage fermentation culture of *Fusarium fujikuroi* involves inoculating a mixed seed culture of *Fusarium fujikuroi* into the fermentation medium, including an early and late fermentation phase. In the early fermentation phase, the culture temperature is 28-30℃ and the aeration rate is 7-8 m³ / h. 3 The stirring speed is 350-450 rpm, and the culture time is 24-48 hours; in the later stage of fermentation, the culture temperature is 31-33℃, and the aeration rate is 5-6 m³ / h. 3 / h, stirring speed 200-350rpm, incubation time 72-96 hours.

[0015] Furthermore, the carbon source in the fermentation medium is one or a combination of starch liquefaction liquid, dextrin, maltose, maltose, lactose, and sucrose.

[0016] Furthermore, the nitrogen source in the fermentation medium is one or a combination of several of the following: soybean meal powder, peanut meal powder, corn protein powder, rice protein powder, dephenolized cottonseed protein, (NH4)2SO4, and NaNO3.

[0017] Compared with existing technologies, this invention provides a method for producing gibberellin GA4+7 mixtures in different proportions through mixed-bacterial fermentation and its applications, offering the following beneficial effects: This invention provides a method for producing gibberellin GA4+7 mixtures in different proportions through mixed-bacterial fermentation. When using a two-stage culture to control fermentation conditions, this mixed strain can produce gibberellin GA4 and GA7 mixtures in different proportions within a short fermentation cycle. After 120 hours of fermentation, the gibberellin GA4+7 mixture has a potency of approximately 1600 mg / L. It has excellent application prospects and plays a positive role in promoting the industrial production of gibberellin GA4+7. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1:

[0020] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF01 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF01 seed solution.

[0021] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF02 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF02 seed solution.

[0022] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed at a ratio of 1:2 (50 mL: 100 mL), resulting in a total volume of 150 mL.

[0023] 150 mL of the mixed and activated seed solution was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of 4.0% glucose, 2.5% soybean meal, 1.5% peanut meal, 2.0% dextrin, 0.15% KH2PO4, 0.03% (NH4)2SO4, and 0.10% MgSO4·7H2O. The fermentation was carried out at 28 °C, with a stirring speed of 300 rpm and an aeration rate of 2.5 m3 / h for 24 hours to obtain the mixed seed solution.

[0024] A 10% (v / v) mixture of seed culture was inoculated into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consisted of: 10.0% starch liquefaction liquid, 1.5% peanut powder, 1.5% soybean powder, 0.12% soybean oil, 0.12% MgSO4·7H2O, 0.04% (NH4)2SO4, 0.18% KH2PO4, and 0.005% ZnSO4. In the early stage of fermentation, the culture temperature was 29℃, the aeration rate was 8 m³ / h, the stirring speed was 450 rpm, and the culture time was 36 hours. In the later stage of fermentation, the culture temperature was 32℃, the aeration rate was 6 m³ / h, the stirring speed was 350 rpm, and the culture time was 84 hours. Fermentation cultured for 120 hours yielded fermentation broth. After testing, the gibberellin GA4 yield was 198 mg / L, the GA7 titer was 1346 mg / L, and the total titer was 1544 mg / L. GA4 accounted for 13% and GA7 accounted for 87%.

[0025] Example 2:

[0026] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF01 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF01 seed solution.

[0027] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF02 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF02 seed solution.

[0028] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed at a ratio of 1:1 (75 mL: 75 mL), resulting in a total volume of 150 mL.

[0029] 150 mL of the mixed and activated seed culture was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of: 4.0% glucose, 2.5% soybean meal, 1.5% peanut meal, 2.0% dextrin, 0.15% KH₂PO₄, 0.03% (NH₄)₂SO₄, and 0.10% MgSO₄·7H₂O. The temperature was 28 °C, the stirring speed was 300 rpm, and the aeration rate was 2.5 m³ / min. 3 Incubate for 24 hours at a time to obtain a mixed seed culture;

[0030] A 10% (v / v) mixture of seed culture was inoculated into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consisted of: 10.0% starch liquefaction liquid, 1.8% peanut powder, 1.5% soybean powder, 0.15% soybean oil, 0.16% MgSO4·7H2O, 0.04% (NH4)2SO4, 0.18% KH2PO4, and 0.005% ZnSO4. In the early stage of fermentation, the culture temperature was 28℃, the aeration rate was 7.5 m³ / h, the stirring speed was 400 rpm, and the culture time was 48 hours. In the later stage of fermentation, the culture temperature was 32℃, the aeration rate was 6 m³ / h, the stirring speed was 300 rpm, and the culture time was 72 hours. Fermentation cultured for 120 hours yielded fermentation broth. After testing, the gibberellin GA4 yield was 363 mg / L, the GA7 titer was 1270 mg / L, and the total titer was 1633 mg / L. GA4 accounted for 22% and GA7 accounted for 78%.

[0031] Example 3:

[0032] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF01 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF01 seed solution.

[0033] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF02 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF02 seed solution.

[0034] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed at a ratio of 2:1 (100mL:50mL), resulting in a total volume of 150mL.

[0035] 150 mL of the mixed and activated seed solution was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of 4.0% glucose, 2.5% soybean meal, 1.5% peanut meal, 2.0% dextrin, 0.15% KH2PO4, 0.03% (NH4)2SO4, and 0.10% MgSO4·7H2O. The fermentation was carried out at 28 °C, with a stirring speed of 300 rpm and an aeration rate of 2.5 m3 / h for 24 hours to obtain the mixed seed solution.

[0036] A 10% (v / v) mixture of seed culture was inoculated into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consisted of: 10.0% dextrin, 2.0% peanut flour, 1.8% soybean flour, 0.10% soybean oil, 0.12% MgSO4·7H2O, 0.04% (NH4)2SO4, 0.18% KH2PO4, and 0.005% ZnSO4. In the early stage of fermentation, the temperature was 29℃, the aeration rate was 8 m³ / h, the stirring speed was 450 rpm, and the culture time was 36 hours. In the later stage of fermentation, the temperature was 32℃, the aeration rate was 6 m³ / h, the stirring speed was 350 rpm, and the culture time was 84 hours. After 120 hours of fermentation, the fermentation broth was obtained. Analysis showed that the gibberellin (GA4) yield was 690 mg / L, the GA7 titer was 946 mg / L, and the total titer was 1636 mg / L. GA4 accounted for 42% and GA7 accounted for 58%.

[0037] Example 4:

[0038] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF01 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF01 seed solution.

[0039] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF02 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF02 seed solution.

[0040] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed at a ratio of 4:1 (120mL:30mL), resulting in a total volume of 150mL.

[0041] 150 mL of the mixed and activated seed solution was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of 4.0% glucose, 2.5% soybean meal, 1.5% peanut meal, 2.0% dextrin, 0.15% KH2PO4, 0.03% (NH4)2SO4, and 0.10% MgSO4·7H2O. The fermentation was carried out at 28 °C, with a stirring speed of 300 rpm and an aeration rate of 2.5 m3 / h for 24 hours to obtain the mixed seed solution.

[0042] A 10% (v / v) mixture of seed culture was inoculated into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consisted of: 10.0% starch liquefaction liquid, 1.5% peanut powder, 1.5% soybean powder, 0.12% soybean oil, 0.12% MgSO4·7H2O, 0.04% (NH4)2SO4, 0.18% KH2PO4, and 0.005% ZnSO4. In the early stage of fermentation, the culture temperature was 29℃, the aeration rate was 8 m³ / h, the stirring speed was 450 rpm, and the culture time was 36 hours. In the later stage of fermentation, the culture temperature was 32℃, the aeration rate was 6 m³ / h, the stirring speed was 350 rpm, and the culture time was 84 hours. Fermentation cultured for 120 hours yielded fermentation broth. After testing, the gibberellin GA4 yield was 1027 mg / L, the GA7 titer was 553 mg / L, and the total titer was 1580 mg / L. GA4 accounted for 65% and GA7 accounted for 35%.

[0043] Example 5:

[0044] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF01 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF01 seed solution.

[0045] Scrape approximately 1 cm long and wide of Fusarium fujikuroi NRF02 mycelium from the slant using an inoculation knife, and inoculate it into a shake flask containing seed culture medium. The seed culture medium consists of 3.0% glucose, 2.0% soybean meal, 1.2% peanut meal, 3.0% dextrin, 0.12% KH2PO4, 0.02% (NH4)2SO4, and 0.10% MgSO4·7H2O. The temperature is 28℃, the shaker speed is 300 rpm, and the culture is carried out for 36 hours to obtain 100 mL of activated Fusarium fujikuroi NRF02 seed solution.

[0046] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed at a ratio of 6.5:1 (130mL:20mL), resulting in a total volume of 150mL.

[0047] 150 mL of the mixed and activated seed solution was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of 4.0% glucose, 2.5% soybean meal, 1.5% peanut meal, 2.0% dextrin, 0.15% KH2PO4, 0.03% (NH4)2SO4, and 0.10% MgSO4·7H2O. The fermentation was carried out at 28 °C, with a stirring speed of 300 rpm and an aeration rate of 2.5 m3 / h for 24 hours to obtain the mixed seed solution.

[0048] The mixed seed culture was inoculated at 10% (v / v) into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consisted of: 10.0% dextrin, 1.8% peanut powder, 2.0% soybean powder, 0.16% soybean oil, 0.12% MgSO4·7H2O, 0.04% (NH4)2SO4, 0.18% KH2PO4, and 0.005% ZnSO4. During the initial fermentation stage, the culture temperature was 28℃ and the aeration rate was 8 m³ / s. 3 The aeration rate was 450 rpm for 48 hours; in the later stage of fermentation, the aeration rate was 33℃, the aeration rate was 6 m³ / h, the aeration rate was 300 rpm, and the fermentation time was 72 hours. After 120 hours of fermentation, the fermentation broth was obtained. Analysis showed that the gibberellin GA4 yield was 1380 mg / L, the GA7 titer was 243 mg / L, and the total titer was 1623 mg / L. GA4 accounted for 85% and GA7 accounted for 15%.

[0049] The first aspect is the cultivation of the mixed seed culture. The specific process is as follows:

[0050] Using an inoculation knife, scrape approximately 1 cm long and wide *Fusarium fujikuroi* NRF01 or NRF02 mycelial blocks from the slant culture. Inoculate these blocks into shake flasks containing seed culture medium. The seed culture medium consists of: glucose 2.0-5.0%, soybean meal 1.0-3.0%, peanut meal 0.8-1.5%, dextrin 2.0-4.0%, KH₂PO₄ 0.08-0.16%, (NH₄)₂SO₄ 0.01-0.03%, and MgSO₄·7H₂O 0.05-0.15%. Incubate at 28-30℃ and 300-400 rpm for 24-48 hours to obtain *Fusarium fujikuroi* NRF01 and NRF02 mycelial blocks, respectively. 50-150 mL of NRF02-activated seed solution;

[0051] The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed in different proportions, and the volume of the mixture was 150 mL.

[0052] 150 mL of the mixed and activated seed solution was inoculated into a 50 L fermenter (30 L / 50 L) containing seed culture medium. The seed culture medium consisted of 2.0-4.0% glucose, 1.5-2.5% soybean meal, 1.0-2.0% peanut meal, 2.0-4.0% dextrin, 0.10-0.20% KH2PO4, 0.02-0.04% (NH4)2SO4, and 0.05-0.15% MgSO4·7H2O. The temperature was 28-30℃, the stirring speed was 250-350 rpm, and the aeration rate was 1.5-3.0 m3 / h. The mixture was cultured for 18-36 hours to obtain the mixed seed solution.

[0053] Secondly, a method for producing a mixture of gibberellin GA4+7 in different proportions through mixed bacterial fermentation involves obtaining a mixed seed culture, followed by staged controlled fermentation conditions. The specific steps are as follows:

[0054] Inoculate the mixed seed culture at 8-12% (V / V) into a 100L fermenter (70L / 100L) containing fermentation medium. The fermentation medium consists of: 5.0%-10.0% carbon source, 2.5-4.0% nitrogen source, 0.08-0.16% soybean oil, 0.08-0.16% MgSO4·7H2O, 0.02-0.05% (NH4)2SO4, 0.15-0.40% KH2PO4, and 0.005-0.01% ZnSO4.

[0055] In the early stage of fermentation, the culture temperature is 28-30℃, the aeration rate is 7-8 m3 / h, the stirring speed is 350-450 rpm, and the culture time is 24-48 hours; in the later stage of fermentation, the culture temperature is 31-33℃, the aeration rate is 5-6 m3 / h, the stirring speed is 200-350 rpm, and the culture time is 72-96 hours.

[0056] Analysis methods for gibberellins GA4 and GA7: Instrument: Shimadzu LC-20AT, using a 150mm × 4.6mm (id) stainless steel column packed with an ODS-C18 (5μm packing) column. Mobile phase: Methanol + water + formic acid = 67 + 33 + 0.05 (v / v), degassed by ultrasonication before use. Flow rate: 0.7 mL / min. Detection wavelength: 210 nm. Injection volume: 20 μL. Sample solution preparation: Adjust the pH of the fermentation broth to approximately 7.0 with 2% NaOH solution, filter with filter paper, accurately pipette 5.0 mL of the filtrate into a 25 mL volumetric flask, dilute to the mark with the mobile phase, centrifuge, collect the supernatant, and filter through a 0.45 μm filter membrane for injection detection. Retention time: approximately 9.4 min for gibberellin GA7, and approximately 11.0 min for gibberellin GA4. A strain producing gibberellin GA4, classified as *Fusarium fujikuroi* NRF01, was deposited at the China Center for Type Culture Collection (CCTCC) on May 19, 2022, with accession number CCTCC NO. M2022683. A strain producing gibberellin GA7, classified as *Fusarium fujikuroi* NRF02, was deposited at the CCTCC on August 1, 2022, with accession number CCTCC NO. M20221211.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0058] In the description of this invention, unless otherwise stated, "a plurality of" means two or more. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0059] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A method for producing a mixture of gibberellin GA4+7 in different proportions by fermentation with mixed bacteria, comprising two high-yield gibberellin strains: one GA4-producing strain, classified as *Fusarium fujikuroi* NRF01, deposited at the China Center for Type Culture Collection (CCTCC) on May 19, 2022, with accession number CCTCC NO. M2022683; and one GA7-producing strain, classified as *Fusarium fujikuroi* NRF02, deposited at the CCTC on August 1, 2022, with accession number CCTCC NO. M20221211. The method is characterized by: Different ratios of high-yielding gibberellin GA4 and GA7 strains were mixed and cultured, with fermentation conditions controlled in stages for 120 hours to obtain mixtures of gibberellin GA4 and GA7 in varying proportions. The staged fermentation conditions included an early fermentation phase and a late fermentation phase. In the early fermentation phase, the culture temperature was 28-30℃ and the aeration rate was 7-8 m³ / h. 3 The stirring speed is 350-450 rpm, and the culture time is 24-48 hours; in the later stage of fermentation, the culture temperature is 31-33℃, and the aeration rate is 5-6 m³ / h. 3 / h, stirring speed 200-350rpm, incubation time 72-96 hours.

2. The method for producing a mixture of gibberellin GA4+7 in different proportions by mixed bacterial fermentation as described in claim 1, characterized in that, Includes the following steps: (1) Using an inoculation knife, scrape approximately 1 cm long and wide *Fusarium fujikuroi* NRF01 or *Fusarium fujikuroi* NRF02 mycelial blocks from the slant culture and inoculate them into shake flasks containing seed culture medium. The seed culture medium consists of: glucose 2.0-5.0%, soybean meal 1.0-3.0%, peanut meal 0.8-1.5%, dextrin 2.0-4.0%, KH2PO4 0.08-0.16%, (NH4)2SO4 0.01-0.03%, MgSO4·7H2O 0.05-0.15%. The temperature is 28-30℃, the shaking speed is 300-400 rpm, and the culture time is 24-48 hours to obtain *Fusarium fujikuroi* NRF01 and *Fusarium* NRF02 mycelial blocks, respectively. 50-150 mL of NRF02-activated seed solution; (2) The activated seed solutions of Fusarium fujikuroi NRF01 and Fusarium fujikuroi NRF02 were mixed in different proportions, and the volume after mixing was 150 mL. (3) Inoculate 150 mL of the mixed and activated seed solution into a 50 L fermenter containing 30 L of seed culture medium. The seed culture medium consists of: glucose 2.0-4.0%, soybean meal 1.5-2.5%, peanut meal 1.0-2.0%, dextrin 2.0-4.0%, KH2PO4 0.10-0.20%, (NH4)2SO4 0.02-0.04%, MgSO4·7H2O 0.05-0.15%, temperature 28-30℃, stirring speed 250-350 rpm, and aeration rate 1.5-3.0 m³ / h. 3 Culture for 18-36 hours per hour to obtain a mixed seed solution; (4) Inoculate 8-12% of the mixed seed culture into a 100L fermenter containing 70L of fermentation medium according to the volume ratio, and carry out two-stage fermentation culture; the composition of the fermentation medium is: carbon source 5.0%-10.0%, nitrogen source 2.5-4.0%, soybean oil: 0.08-0.16%, MgSO4·7H2O: 0.08-0.16%, (NH4)2SO4: 0.02-0.05%, KH2PO4: 0.02-0.05%. 4： 0.15-0.40%, ZnSO 4： 0.005-0.01%; the two-stage fermentation process includes an early fermentation stage and a late fermentation stage; in the early fermentation stage, the culture temperature is 28-30℃ and the aeration rate is 7-8 m³ / h. 3 The stirring speed is 350-450 rpm, and the culture time is 24-48 hours; in the later stage of fermentation, the culture temperature is 31-33℃, and the aeration rate is 5-6 m³ / h. 3 / h, stirring speed 200-350rpm, incubation time 72-96 hours.

3. The method for producing a mixture of gibberellin GA4+7 in different proportions by mixed bacterial fermentation as described in claim 2, characterized in that, The carbon source in the fermentation medium is one or a combination of starch liquefaction liquid, dextrin, maltose, maltose, lactose, and sucrose.

4. The method for producing a mixture of gibberellin GA4+7 in different proportions by mixed bacterial fermentation as described in claim 2, characterized in that, The nitrogen source in the fermentation medium is one or a combination of several of the following: soybean meal powder, peanut meal powder, corn protein powder, rice protein powder, dephenolized cottonseed protein, (NH4)2SO4, and NaNO3.

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