Engineering bacterium for high-yield production of gibberellic acid GA4+7 as well as construction method and application thereof

Abstract

The invention provides a high-yield gibberellic acid GA4 + 7 engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of gene engineering. According to the invention, gibberella zeylanica is taken as an original strain, and a P450-3 gene is knocked out by utilizing a CRISPR-Cas9 gene editing technology, so that a strain which does not generate GA1 and GA3 is obtained, and the total yield of GA4 + 7 is greatly increased; according to the present invention, the DES dehydrogenase is subjected to key amino acid mutation, the selected mutation sites are T50A, G108P and E126K, the combining ability of the mutated DES dehydrogenase and GA4 is enhanced, and the mutated DES dehydrogenase has strong catalytic ability under high temperature and acidic fermentation conditions so as to adjust the ratio of GA4 + 7 in the fermentation broth, such that the total amount of GA4 + 7 is increased by 314.95%;

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to high-yield gibberellic acid (GA) production. 4+7 Engineered bacteria, their construction methods, and applications. Background Technology

[0002] Gibberellins (GAs), also known as gibberellins, are important plant growth hormones belonging to the tetracyclic diterpenoid class of compounds. They are diverse and can be classified into 20C-GA and 19C-GA based on the number of carbon atoms they contain. Currently, 136 types of gibberellins have been discovered, collectively known as gibberellins (GAs). They are systematically numbered GA1-GA according to the order of their discovery. 136 The GAs that were preferentially discovered generally had high activity, such as GA1, GA3, GA4, and GA7. Since 1950, researchers have devoted a great deal of time to studying the synthesis process of gibberellins in plants and microorganisms, and have discovered many new gibberellin varieties. GA4 and GA7, due to their similar properties and the difficulty in separation and purification, often exist as a mixture, namely GA4+GA7. The high activity and unique advantages of GA4+GA7 have attracted widespread attention.

[0003] Gibberellins, as plant growth regulators, have various regulatory functions. The amount of gibberellins produced varies 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 is found in the roots, stems, leaves, and seeds. In terms of promoting stem growth, the 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 epidermal cell growth, resulting in a thinner cuticle and making fruits prone to spotting and cracking. GA7 strongly inhibits flower bud formation, while GA4 not only does not inhibit it but actually promotes it. The combination of GA4 and GA7 offers moderate activity and can complement each other. Furthermore, GA4 + GA7 can break dormancy without inducing hypocotyl growth, resulting in good flowering, high fruit set, improved fruit cuticle toughness, and prevention of brown spot disease. Curry applied 15 mg / L and 30 mg / L GA to apples. 4+7Afterwards, the epidermal cell density of the fruit increased by 14% and 27%, respectively, and the incidence of brown spot disease decreased by 40% and 83%, respectively (Curry E. Increase in epidermal planar cell density ac-companies decreased russeting of 'golden delicious' apples treated with gibberellin As 4+7 [J]. Hort Science, 2012, 47(2): 232-237.). Kim et al. found that GA4+GA7 can effectively prolong the wilting time of flowers and leaves (Kim HJ, Miller W B. GA...). 4+7 plus BA enhances post-production quality in pot tulips[J].Postharvest Biologyand Technology,2009,51(2):272-277.).

[0004] Currently, gibberellin can be obtained through three methods: chemical synthesis, plant extraction, and microbial fermentation. Because the first two methods are costly and inefficient, while microbial fermentation is quick and efficient, it is widely used in large-scale industrial production. *Gibberella fuciformis* (Fujikura gibberellin) Gibberella fujikuroi GA4 is currently the main strain for industrial production of gibberellic acid, but the yield of GA4+GA7 is still relatively low, making it difficult to meet the needs of industrial production. Further improvements are needed to achieve higher yields. In addition, field experience shows that a GA4:GA7 ratio of about 2:1 is the most effective ratio; however, the GA4:GA7 produced by existing strains does not meet this ratio and does not meet the needs of actual field production. Summary of the Invention

[0005] In view of this, the present invention provides an engineered strain that produces high levels of gibberellic acid GA4+GA7, its construction method, and its applications, thereby solving the problem of GA4+GA7 production by wild-grown *Fujikura fujikuro*. 4+7 The problem is that the yield is low and the proportions do not match those used in field production.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides high-yield gibberellic acid (GA) production. 4+7 Engineered bacteria, including the starting strain, P450-3 Gene knockout plasmids and dehydrogenases DES Gene mutation plasmid; the starting strain is Fumigranobacter fusiforme.

[0007] Preferably, the *Fusarium graminearum* strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.11101.

[0008] This invention also provides a method for constructing the engineered strain that produces high levels of gibberellic acid (GA4+7), comprising the following steps: (1) Using Fusarium graminearum genomic DNA as a template, amplification was performed separately. P450-3 The homologous upstream and homologous downstream sequences at both ends of the gene are extracted, and the homologous upstream and homologous downstream sequences are fused to obtain a fusion fragment; (2) Plasmids containing sgRNA backbone pUC57-sgRNA Using P4503-sgRNA-F1 / R1 as a template, PCR amplification was performed to obtain the sgRNA expression cassette. (3) Link the sgRNA expression cassette into pAN7-Cas9 In plasmids, knockout is formed P450-3 The gene editing plasmid is named pAN7-Cas9-P4503 Its nucleotide sequence is shown in SEQ ID NO.19; (4) The constructed gene-editing plasmid and fusion fragment were transformed into Fusarium oxysporum, and positive clones were screened to obtain knockout clones. P450-3 Fusarium graminearum of the gene; (5) DES The gene is mutated so that nucleotides 148-150 are replaced by GCC instead of ACT, nucleotides 322-324 are replaced by CCT instead of GGA instead of GCT, and nucleotides 376-378 are replaced by AAG instead of GAG. (5) The mutated DES Genes are fused into eukaryotic expression cassettes containing promoters and terminators to obtain... ptrpC::DES-ttrpC Module; (6) ptrpC::DES-ttrpC Module connected to pAN7-1 Dehydrogenase was obtained from the plasmid. DES Gene mutation plasmid, named pAN7-1-ptrpC::DES-ttrpC ; (7) Dehydrogenase DES Transformation of gene mutant plasmids into knockout P450-3 An engineered strain that produces high levels of gibberellic acid GA4+7 was obtained from the *Fujikura fusarium* strain.

[0009] Preferably, the nucleotide sequence of the fusion fragment is shown in SEQ ID NO.11.

[0010] Preferably, the nucleotide sequence of the primer pair P4503-sgRNA-F1 / R1 is shown in SEQ ID NO.15 and SEQ ID NO.16.

[0011] Preferred, mutated DES The protein expressed by the gene has been mutated at amino acid positions 50, 108 and 126. The sequence of the mutated DES protein is shown in SEQ ID NO.30.

[0012] Preferably, the promoter is derived from a plasmid. pFC332 of trpC The promoter, the terminator is derived from plasmid pAN7-1 of trpC Termination of contract.

[0013] The present invention also provides the application of the engineered bacteria in the microbial fermentation preparation of gibberellin GA4+7.

[0014] This invention also provides the application of the engineered bacteria obtained by the above construction method in the microbial fermentation preparation of gibberellin GA4+7.

[0015] Preferably, the engineered bacterial strain is inoculated into a fermentation medium and cultured for 7 days at 28 ℃ and 250 rpm. The fermentation medium consists of: 75 g / L corn starch, 43.5 g / L sucrose, 5 g / L soybean meal, 5 g / L peanut powder, 0.5 g / L KH2PO4, 0.5 g / L K2SO4, and 0.11 g / L MgSO4·7H2O, with water as the solvent.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: 1. P450-3 The gene-encoded P450-3 monooxygenase catalyzes the conversion of GA4 to GA1 and GA7 to GA3 in the gibberellic acid metabolic pathway. This invention utilizes knockout... P450-3 By modifying the gene, strains that do not produce GA1 and GA3 were obtained, resulting in a significant increase in the total yield of GA4 and GA7.

[0017] 2. DES dehydrogenase can catalyze the formation of GA7 from GA4. In this invention, key amino acid mutations were performed on DES dehydrogenase. The selected mutation sites were T50A, G108P, and E126K. The mutated DES dehydrogenase has enhanced binding ability with GA4 and has stronger catalytic ability under high temperature and acidic fermentation conditions, thereby regulating the ratio of GA4 and GA7 in the fermentation broth.

[0018] 3. This invention achieves this by knocking out... P450-3 Genes, and overexpression of the modified gene DESThe gene has increased the total amount of GA4+7 by 314.95% and optimized the ratio of GA4 and GA7, significantly reducing the production cost of GA4 and GA7 and making them more suitable for field needs, eliminating the need for complicated GA4 and GA7 ratio configuration. Attached Figure Description

[0019] Figure 1 This is a single colony of the engineered bacteria that produces high levels of gibberellic acid GA4 and GA7 according to the present invention.

[0020] Figure 2 Map of the pAN7-Cas9 gene editing vector.

[0021] Figure 3 For the starting strain, knockout P450-3 Strains of the gene and simultaneous knockout P450-3 Genes, mutations DES The strain that produces GA 4+7 The curve graph. Detailed Implementation

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] In the embodiments of the present invention pFC332 plasmids and pAN7-1 The plasmids were commercially available and were purchased from Wuhan Gray Algae Biotechnology Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd., respectively.

[0024] Example 1. P450-3 gene knockout The starting strain of this invention is *Fujikura fusiforme* (… Fusarium fujikuroi QJGA4-1 was deposited at the China General Microbiological Culture Collection Center on July 15, 2015, with accession number CGMCC NO.11101; the deposit address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] Step 1. P450-3 Amplification of upstream and downstream homologous sequences of a gene Using Fumigranorhizium anisopliae genomic DNA as a template, amplification was performed separately. P450-3 Homologous upstream and downstream sequences approximately 500 bp from both ends of the gene (GenBank ID: FFUJ_143376).

[0026] The primers for amplifying the homologous upstream sequence are: P4503-UP-F1: GCACTCCCGCAAAGAAATTCAACA (SEQ ID NO. 1); P4503-UP-R1:TCATAGTTAAAAGAAATTTTAGGATCGTAC (SEQ ID NO.2); The homologous downstream amplification primers are: P4503-DW-F1:ATACAGTGGCGGTATGAAATGGG (SEQ ID NO.3); P4503-DW-R1: GACAGATGGGAAGTGGGAGGATTTAC (SEQ ID NO. 4).

[0027] The PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2. Table 1 PCR amplification system

[0028] Table 2 PCR amplification program

[0029] The homologous upstream sequence is: GCACTCCCGCAAAGAAATTCAACACTCGGCCTAAAATTCTCTTTTCAGCAAGTCAGGCAACTCAATTTGACCATCGTGTGCCATTGTACTAATCAAAACTTCGTCACCGTCTGCGTCGGTGGTGTAGGA TCTCGTATTCAAGCCGTGTCCTCTAATGCATGACGTATAGTGACAATCGCGAGACCAGAGAACAATCTATAGAACAATGTCACGTTGCATATTGTGATATCTTTTGTTCCCGAGGCTACTACATGCACAC CTAGCGCCTAAGTTCTGTTTCGGGGTTACTAGCCTATCTCCGTGCCATCACAGCGGCTGAATGTGAATACCGATGCATGGGACCTTAGCGCGGACTGGCGACGTCTGAGGACAGCTGGTATGGATCTAG AATTACCTTTGTCATTTTTGTCATTTATGCTAGATGTCTCCGATAGGGAAACAATATTGTTCCGTTACTAACCATTTACCCTTTGAAGATTCATCAGTACGATCCTAAAATTTCTTTTAACTATGA (SEQ IDNO.5). Homologous downstream sequences are: (SEQ ID NO.6). Step 2. Fuse the homologous upstream and homologous downstream sequences. (1) Add the adapter required for fusion PCR between the homologous upstream sequence and the homologous downstream sequence. Using the amplified homologous upstream sequence as a template, and with P4503-UP-F1 and P4503-UP-R2 as primers, the homologous upstream sequence with adapter was amplified. The amplification system and amplification procedure were the same as above.

[0030] P4503-UP-F1: GCACTCCCGCAAAGAAATTCAACA (SEQ ID NO. 1); P4503-UP-R2: ACCGCCACTGTAT TCATAGTTAAAAGAAATTTTAGGATCGTAC (SEQ ID NO.7) (The underlined part is the connector sequence).

[0031] Using the amplified homologous downstream sequence as a template, and with P4503-DW-F2 and P4503-DW-R1 as primers, amplification was performed to amplify the homologous downstream sequence with adapter. The amplification system and amplification procedure were the same as above.

[0032] P4503-DW-F2: CTTTTAACTATGA ATACAGTGGCGGTATGAAATGGG (SEQ ID NO.8); P4503-DW-R1: GACAGATGGGAAGTGGGAGGATTTAC (SEQ ID NO.4).

[0033] The homologous upstream sequence with adaptor is: GCACTCCCGCAAAGAAATTCAACACTCGGCCTAAAATTCTCTTTTCAGCAAGTCAGGCAACTCAATTTGACCATCGTGTGCCATTGTACTAATCAAAACTTCGTCACCGTCTGCGTCGGTGGTGTAGGATCTCGTATTCAAGCCGTGTCCTCTAATGCATGACGTATAGTGACAATCGCGAGACCAGAGAACAATCTATAGAACAATGTCACGTTGCATATTGTGATATCTTTTGTTCCCGAGGCTACTACATGCACACCTAGCGCCTAAGTTCTGTTTCGGGGTTACTAGCCTATCTCCGTGCCATCACAGCGGCTGAATGTGAATACCGATGCATGGGACCTTAGCGCGGACTGGCGACGTCTGAGGACAGCTGGTATGGATCTAGAATTACCTTTGTCATTTTTGTCATTTATGCTAGATGTCTCCGATAGGGAAACAATATTGTTCCGTTACTAACCATTTACCCTTTGAAGATTCATCAGTACGATCCTAAAATTTCTTTTAACTATGAATACAGTGGCGGT (SEQ ID NO.9). The homologous downstream sequence with adaptor is: (SEQ ID NO.10). (2) The homologous upstream and downstream sequence fragments with adapters in (1) are fused. The fusion primers are P4503-UP-F1 and P4503-DW-R1. The fusion PCR is performed in two steps.

[0034] P4503-UP-F1: GCACTCCCGCAAAGAAATTCAACA (SEQ ID NO. 1); P4503-DW-R1: GACAGATGGGAAGTGGGAGGATTTAC (SEQ ID NO. 4); The reaction system for the first round of fusion PCR is shown in Table 3, and the reaction procedure for the first round of fusion PCR is shown in Table 4; the reaction system for the second round of fusion PCR is shown in Table 5, and the reaction procedure for the second round of fusion PCR is shown in Table 2. Table 3. Fusion PCR One-Round Reaction System

[0035] Table 4. One-round reaction procedure for fusion PCR

[0036] Table 5. Fusion PCR Second Round Reaction System

[0037] The fused fragment sequence is as follows: Step 3. Construction of sgRNA expression cassette Using TCAGCAATCCACATGTCGG (SEQ ID NO.12) (Genomic location P450-3: 1224-1233) and TGGAGAAGAACCTCATCAAG (SEQ ID NO.13) (Genomic location P450-3: 191-210) as target sequences, an sgRNA expression cassette was constructed to target... P450-3 Genes are knocked out.

[0038] Using the plasmid pUC57-sgRNA containing the sgRNA backbone, which was constructed in the laboratory beforehand, as a template, PCR amplification was performed using P4503-sgRNA-F1 / R1 primers. The PCR amplification system and procedure were the same as in "Step 1", and the amplification product was the sgRNA expression cassette.

[0039] The pUC57-sgRNA plasmid sequence is shown in SEQ ID NO.14: P4503-sgRNA-F1: cgaatccccgctgttgtatg TCACGCAATCCACATGTCGGGTTTTAGAGCTAGAAATAG (SEQ IDNO.15); P4503-sgRNA-R1: CTATTTCTAGCTCTAAAAC CTTGATGAGGTTCTTCTCCACGAGCTTCTGCAGAGAACTGC (SEQ ID NO. 16); The underlined sequences are the adapter sequences required for ligation onto the plasmid, and the bolded sequences are the sgRNA sequences.

[0040] The amplified sgRNA expression cassette sequence is shown in SEQ ID NO.17. (SEQ ID NO.17). Step 4. Linking the sgRNA expression cassette to the vector The sgRNA expression cassette was ligated into a pre-constructed [system / feature] in our laboratory. pAN7-Cas9 Within the plasmid, a complete knockout is formed. P450-3 A gene-editing plasmid containing hygromycin resistance. pAN7-Cas9 The plasmid is pre-designed with restriction enzyme sites for inserting sgRNA expression cassettes. pAN7-Cas9 The plasmid was digested with Spe I, and inserted into the sgRNA expression cassette using a one-step cloning kit (Novizan ClonExpress II One Step Cloning Kit-C112) according to the manufacturer's instructions. The ligation product was transformed into competent E. coli cells, and after sequencing confirmation, the cells were stored as knockout cells. P450-3 The gene editing plasmid is named pAN7-Cas9-P4503 .

[0041] pAN7-Cas9 The plasmid sequence is shown in SEQ ID NO.18. pAN7-Cas9-P4503 The plasmid sequence is shown in SEQ ID NO.19: Step 5. Transformation of Fusarium oxysporum with recombinant plasmids into Gibberella fuciformis. The constructed gene-editing plasmid pAN7-Cas9-P4503 The fusion fragment was transformed into *Fujikura fusarium* using electroporation. The specific steps are as follows: 1) Suspend the protoplasts of *Gibberella fuciformis* in 1 M sorbitol solution A, count them under a microscope, and adjust the protoplast concentration of the protoplast solution to 1 × 10⁻⁶. 7 cells / mL; 2) Exogenous plasmids used for transformation pAN7-Cas9-P4503 After purification, the plasmid and fusion fragment were dissolved in sterile deionized water to make the final concentration of the mixed solution of plasmid and fusion fragment 1 μg / μL. 10 μg of the mixed solution was added to every 100 μL of protoplast fluid, mixed well, and incubated on ice for 30 min to form a mixed solution. 3) Add 40 μL of the mixture obtained in step 2) to the pre-cooled 2 cm electroporation cup, then incubate on ice for 5 min. Prepare the electroporation apparatus and set the electroporation parameters as follows: voltage 850 V, capacitance 25 μF, resistance 250 Ω. Perform electroporation. 4) After electroporation, add 1 mL of 1 M sorbitol solution B to the electroporation vessel, incubate on ice for 0.5-1 h, suspend, and spread onto MYG solid regeneration plates, 200 μL / plate. Incubate at 28 ℃ for 12 h, then cover the regeneration plates with soft agar MYG regeneration medium containing hygromycin at a final concentration of 50 μg / mL, 10 mL / plate, and incubate at 28 ℃ until transformants appear; the sorbitol solution B contains 10 mM Tris-Cl, 50 mM CaCl2, and pH 7.5; 5) Transformants were subcultured on soft agar MYG regeneration medium containing hygromycin at a final concentration of 100 μg / mL, and stored after stable growth.

[0042] After overnight resuscitation, the culture was carried out on hygromycin-resistant plates at 28 °C until colonies grew. The grown colonies were then cultured for three generations on PDA plates containing hygromycin resistance. DNA was extracted for identification, and correctly identified strains were preserved to obtain positive transformants. Δp450-3 strains.

[0043] Example 2. Mutation of key amino acids in DES dehydrogenase Step 1. DES Genes and pTOPO Carrier connection Using cDNA from *Fujikura fusiforme* (the starting strain) as a template and DES-F1 / R1 as primers, the enzyme encoding DES dehydrogenase was amplified. DES The gene sequence (GenBank number: FFUJ_14331), amplification system and procedure are the same as step 1 in Example 1.

[0044] DES-F1: ATGCCTCATAAAGATAATCTTCTTGAATCGC (SEQ ID NO. 20); DES-R1: CTACCAGAATGCAATGAACTTGGTC (SEQ ID NO. 21); DES The sequence of the gene amplification fragment is as follows: Using the zero-background pTOPO-Blunt Simple blunt-end cloning kit from Beijing Aidlab Biotechnology Co., Ltd., and following the instructions (http: / / www.aidlab.cn / products-show.asp?anclassid=91&nclassid=70&id=1841), the amplified... DES Fragments and pTOPO The vector (provided in the kit) is used for ligation, and the ligation product is... pTOPO-DES .

[0045] Step 2. Design mutation sites The unmutated DES protein sequence is as follows: MPHKDNLLESPVGKSVTATIAYHSGPALPTSPIAGVTTLQDCTQQAVAVTDIRPSVSSFTLDGNGFQVVKHTSAVGSPPYDHSSWTDPVVRKEVYDPEIIELAKSLTGAKKVMILLASSRNVPFKEPELAPPYPMPGKSSSGSKEREAIPANELPTTRAKGFQKGEEEGPVRK PHKDWGPSGAWNTLRNWSQELIDEAGDIIKAGDEAAKLPGGRAKNYQGRRWALYTTWRPLKTVKRDPMAYVDYWTADEEDGVSFWRNPPGVHGTFESDVLLTKANPKHKWYWISDQTPDEVLLMKIMDTESEKDGSEIAGGVHHCSFHLPGTEKEEVRESIETKFIAFW* (SEQ ID NO.23). The DES protein sequence was analyzed, and point mutations were performed at amino acids 50, 108, and 126. ​ Nucleotides 148-150 of the gene are replaced by ACT with GCC (T50A), nucleotides 322-324 are replaced by GGA with CCT (G108P), and nucleotides 376-378 are replaced by GAG with AAG (E126K).

[0046] The specific steps are as follows: According to the design ​ The mutation sites of the gene were identified, and corresponding point mutation primers were designed to complete the mutation of key amino acids in DES dehydrogenase. The designed point mutation primers are as follows: DES-T50A-F1: TAGCAGTGgccGATATCCGCCCTTCAGTCTCG (SEQ ID NO. 24); DES-T50A-R1: GATATCggcCACTGCTACGGCCTGCTGAGTGC (SEQ ID NO. 25); DES-G108P-F1: TCTCACTcctGCCAAGAAGGTCATGATTCTACTTG (SEQ ID NO. 26); DES-G108P-R1:TCTTGGCaggAGTGAGAGACTTTGCCAGTTCAATG (SEQ ID NO. 27); DES-E126K-F1: CTTCAAGaagCCAGAGCTCGCCCCTCCTTATC (SEQ ID NO. 28); DES-E126K-R1: GCTCTGGcttCTTGAAGGGAACATTCCGAGAC (SEQ ID NO. 29).

[0047] In step 1 ​ Using DES-T50A-F1 and DES-T50A-R1 as templates, a first round of point mutation was performed. The PCR amplification system and procedure were the same as step 1 in Example 1. The PCR product was transformed into competent E. coli cells, and after sequencing confirmation, the first round of point mutation was completed. Subsequent rounds of point mutation were performed using the previous round's point mutation product as a template, employing the corresponding point mutation primers, until three rounds of point mutation were completed.

[0048] The mutated DES protein sequence is as follows: MPHKDNLLESPVGKSVTATIAYHSGPALPTSPIAGVTTLQDCTQQAVAVADIRPSVSSFTLDGNGFQVVKHTSAVGSPPYDHSSWTDPVVRKEVYDPEIIELAKSLTPAKKVMILLASSRNVPFKKPELAPPYPMPGKSSSGSKEREAIPANELPTTRAKGFQKGEEEGPVRK PHKDWGPSGAWNTLRNWSQELIDEAGDIIKAGDEAAKLPGGRAKNYQGRRWALYTTWRPLKTVKRDPMAYVDYWTADEEDGVSFWRNPPGVHGTFESDVLLTKANPKHKWYWISDQTPDEVLLMKIMDTESEKDGSEIAGGVHHCSFHLPGTEKEEVRESIETKFIAFW* (SEQ ID NO.30).

[0049] Step 3. The mutated... ​ Gene insertion​ In the overexpression module After mutation ​ Gene insertion ​ In the overexpression module, the following primers were designed: ptrpC-F1:GGAGGTCAACACATCAATGCTTATTTTG (SEQ ID NO. 31); ptrpC-R1: CTTTATGAGGCATGAATTCGATCAAGGCTTGGG (SEQ ID NO. 32); DES-F2: CTTGATCGAATTCATGCCTCATAAAGATAATCTTCTTGAATCG (SEQ ID NO.33); DES-R2: TCGCGTATTGTCGCTGTGCActaCCAGAATGCAATGAACTTGGTCTC (SEQ ID NO.34); ttrpC-F1: TGCACAGCGACAATACGCGAggatccacttaacgttatactgaaa (SEQ ID NO. 35); ttrpC-R1:tcgagtggagatgtggagtggg (SEQ ID NO. 36). Using commercial plasmids ​ After mutation ​ Sequences and commercial plasmids ​ Using templates, amplification ​ promoter, with connector ​ sequence sum ​ The terminator, PCR amplification system, and procedure are the same as step 1 in Example 1. ​ The promoter amplification template is a commercial plasmid. ​ Purchased from Wuhan Gray Algae Biotechnology Co., Ltd. ​ The amplification template for the terminator is a commercial plasmid. ​ Purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0050] The amplification sequence of primer ptrpC-F1 / R1 is as follows: GGAGGTCAACACATCAATGCTTATTTTGGTTTAGTCGTCCAGGCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGTAGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTGTCATCTGCTGCTTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGCCCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCCAAGCCTTGATCGAATTCATGCCTCATAAAG (SEQ ID NO.37). The amplified sequence of primer DES-F2 / R2 is: The amplification sequence of primer ttrpC-F1 / R1 is as follows: (SEQ ID NO.39). The three fragments were fused using fusion PCR, including two rounds of fusion PCR reactions. The fusion primers were ptrpC-F1 and ttrpC-R1. The reaction system for the first round of fusion PCR is shown in Table 6, and the reaction system for the second round of fusion PCR is shown in Table 7. The reaction programs for the first and second rounds of fusion PCR are shown in Table 4 and Table 2, respectively.

[0051] Table 6. Fusion PCR One-Round Reaction System

[0052] Table 7 Fusion PCR Second Round Reaction System

[0053] Fusion to obtain eukaryotic expression cassettes ​ Module for fusing PCR products ​ ​ Using the template, the following primers were designed for amplification to introduce the adapter sequence.

[0054] ptrpC-F2: ​ GGAGGTCAACACATCAATGCTTATTTTG (SEQ ID NO. 40); ttrpC-R2: ​ tcgagtggagatgtggagtggg (SEQ ID NO. 41); The underlined sequences are the adapter sequences required to connect to the plasmid.

[0055] Using primers pAN7-F1 / R1, PCR was performed to... ​ Linearize the plasmid vector (hygromycin resistance), and follow the same PCR amplification system and procedure as above.

[0056] pAN7-F1:GAATTCCCTTGTATTCCTACAC (SEQ ID NO. 42); pAN7-R1: GTAATCATGGTCATAGCTGTTT (SEQ ID NO. 43); Using the one-step cloning kit (Novizan ClonExpress II One Step Cloning Kit-C112), follow the instructions to... ​ Module connected to ​ A complete expression plasmid is formed within the plasmid, which is then transformed into competent E. coli cells. After successful sequencing, the cells are transferred to a knockout plasmid. P450-3 The Fujikura Fuciformis gene was revived overnight, then inverted into a resistant plate and cultured at 28°C until colonies grew.

[0057] The grown colonies were cultured for three generations on PDA plates containing hygromycin resistance. DNA was extracted for identification, and after successful sequencing, the correctly identified strains were preserved. ​ strains.

[0058] ​ The plasmid sequence is: Example 3. Gibberellin metabolism experiment Using a sterile inoculation spatula, the positive transformants from Example 1, which had been verified through rescreening, were... ​ The positive transformant Δ from Example 2, which was verified through rescreening, ​ The cultures were separately inoculated into sterilized seed culture medium (seed culture medium: corn starch 20 g / L, sucrose 15 g / L, peanut meal 15 g / L, soybean meal 3 g / L, KH2PO4 1 g / L, MgSO4 1 g / L, solvent: water), and cultured on a shaker at 28℃ and 250 rpm for 2 days. Then, the cultures were transferred to fermentation medium (fermentation medium: corn starch 75 g / L, sucrose 43.5 g / L, soybean meal 5 g / L, peanut meal 5 g / L, KH2PO4 0.5 g / L, K2SO4 0.5 g / L, MgSO4·7H2O 0.11 g / L, solvent: water) at an inoculum size of 6%. After culturing on a shaker at 28℃ and 250 rpm for 7 days, the supernatant was collected, diluted, and the yields of GA3, GA4, and GA7 were determined by high-performance liquid chromatography (HPLC). Wild *Fujikura scab* was used as a control. Each group was set up in triplicate. The results are shown in the figure. ​ See Table 8.

[0059] Table 8. Comparison of yields among groups after 120 h of cultivation.

[0060] As shown in Table 8, ​ Engineered bacteria not only improved GA 4+7 The yield was adjusted, and the ratio of GA4 to GA7 was modified to better suit the needs of field production.

[0061] As can be seen from the above embodiments, the present invention provides a high-yield gibberellic acid (GA) production method. 4+7 The present invention describes the engineered bacteria, its construction method, and its application. By directionally modifying wild Fusarium oxysporum, a strain that does not produce GA1 and GA3 was obtained, thereby significantly increasing the total yield of GA4+GA7. The ratio of GA4 and GA7 was also adjusted to meet production requirements.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered bacterium that produces high levels of gibberellic acid GA4+7, characterized in that, Including the starting strain, P450-3 Gene knockout plasmids and dehydrogenases DES Gene mutation plasmid; the starting strain is Fumigranobacter fusiforme.

2. The engineered bacteria according to claim 1, characterized in that, The *Fujikura scab* strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.11101.

3. The high-yield gibberellic acid (GA) production method according to claim 1 4+7 The method for constructing engineered bacteria is characterized by, Includes the following steps: (1) Using Fusarium graminearum genomic DNA as a template, amplification was performed separately. P450-3 The homologous upstream and homologous downstream sequences at both ends of the gene are extracted, and the homologous upstream and homologous downstream sequences are fused to obtain a fusion fragment; (2) Using plasmids containing the sgRNA backbone pUC57-sgRNA Using P4503-sgRNA-F1 / R1 as a template, PCR amplification was performed to obtain the sgRNA expression cassette. (3) Link the sgRNA expression cassette into pAN7-Cas9 In plasmids, knockout is formed P450-3 The gene editing plasmid is named pAN7-Cas9-P4503, Its nucleotide sequence is shown in SEQ ID NO.19; (4) The constructed gene-editing plasmid and fusion fragment were transformed into Fusarium oxysporum, and positive clones were screened to obtain knockout clones. P450-3 Fusarium graminearum of the gene; (5) DES The gene is mutated so that nucleotides 148-150 are replaced by GCC instead of ACT, nucleotides 322-324 are replaced by CCT instead of GGA instead of GCT, and nucleotides 376-378 are replaced by AAG instead of GAG. (5) The mutated DES Genes are fused into eukaryotic expression cassettes containing promoters and terminators to obtain... ptrpC::DES-ttrpC Module; (6) ptrpC::DES-ttrpC Module connected to pAN7-1 The dehydrogenase encoding gene was obtained from the plasmid. DES The mutant plasmid was named pAN7-1-ptrpC::DES-ttrpC ; (7) Dehydrogenase DES Transformation of gene mutant plasmids into knockout P450-3 An engineered strain that produces high levels of gibberellic acid GA4+7 was obtained from the *Fujikura fusarium* strain.

4. The construction method according to claim 3, characterized in that, The nucleotide sequence of the fusion fragment is shown in SEQ ID NO.

11.

5. The construction method according to claim 3, characterized in that, The nucleotide sequences of the primer pair P4503-sgRNA-F1 / R1 are shown in SEQ ID NO.15 and SEQ ID NO.

16.

6. The construction method according to claim 3, characterized in that, After mutation DES The protein expressed by the gene has been mutated at amino acid positions 50, 108 and 126. The amino acid sequence of the mutated DES protein is shown in SEQ ID NO.

30.

7. The construction method according to claim 3, characterized in that, The promoter is derived from a plasmid. pFC332 of trpC The promoter, the terminator is derived from plasmid pAN7-1 of trpC Termination of contract.

8. The application of the engineered bacteria according to claim 1 or 2 in the microbial fermentation preparation of gibberellin GA4+7.

9. The application of engineered bacteria constructed by the construction method according to any one of claims 3 to 7 in the microbial fermentation preparation of gibberellin GA4+7.

10. The application according to claim 8 or claim 9, characterized in that, The engineered bacterial strain was inoculated into a fermentation medium and cultured for 7 days at 28°C and 250 rpm. The fermentation medium consisted of: 75 g / L corn starch, 43.5 g / L sucrose, 5 g / L soybean meal, 5 g / L peanut powder, 0.5 g / L KH2PO4, 0.5 g / L K2SO4, and 0.11 g / L MgSO4·7H2O, with water as the solvent.

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