CN114517161B

CN114517161B - High yield gibberellin GA3Genetically engineered bacterium of (2), construction method and application

Info

Publication number: CN114517161B
Application number: CN202210232658.XA
Authority: CN
Inventors: 柳志强; 王浩南; 柯霞; 黄良刚; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-03-10
Filing date: 2022-03-10
Publication date: 2024-04-19
Anticipated expiration: 2042-03-10
Also published as: CN114517161A

Abstract

The invention relates to a genetically engineered bacterium for high-yield gibberellin GA ₃, a construction method and application thereof. The invention enhances the forward regulation and control action of the gene expressing the nitrogen source regulation factor AreA and the gene expressing the global regulation factor Lae1 in the gibberella, enhances the transcription level of the related genes and enhances the accumulation of gibberellin GA ₃. The recombinant gibberella caput-medusa of the high-yield gibberellin GA ₃ provided by the invention has the advantages that compared with a wild type, the recombinant gibberella caput-medusa has the advantages that the overall transcription level of GA gene clusters is enhanced, the synthesis capacity of metabolic processes is improved, the transcription inhibition of GA ₃ synthesis related genes is weakened, the carbon source substance and the nitrogen source substance can be better utilized for producing gibberellin GA ₃, the level of the strain with the optimal performance after transformation for fermenting to produce gibberellin GA ₃ is improved to 3.25 g/L from 2 g/L compared with the original strain, the production capacity of gibberellin GA ₃ is obviously improved, and the recombinant gibberella caput-medusa can be applied to the production of large-scale gibberellin GA ₃.

Description

Genetically engineered bacterium for high yield of gibberellin GA 3, construction method and application

Technical Field

The invention relates to a genetically engineered bacterium for high-yield gibberellin GA ₃, a construction method and application thereof.

Background

Gibberellin Acid (GA ₃) is a plant growth hormone synthesized by plants and partial fungi. Because of its special ability to stimulate plant growth, it is widely used in agriculture, horticulture and wine production. The trichoderma reesei was first discovered by a plant pathologist in japan in studying rice bakanae disease, and subsequent studies confirm that the trichoderma reesei naturally has complete GA ₃ synthesis ability. GA ₃ is tetracyclic diterpenoid carboxylic acid, the molecular formula is C ₁₉H₂₂O₆, the molecular weight is 346.37, the melting point is 233-235 ℃, and the tetracyclic diterpenoid carboxylic acid is easy to dissolve in alcohols, acetone, ethyl acetate, sodium bicarbonate solution and phosphate buffer with pH of 6.2, and is difficult to dissolve in water and diethyl ether. GA ₃ is generally obtained as a secondary metabolite by means of liquid fermentation, solid fermentation and plant extraction. Currently, GA ₃ production mainly depends on liquid submerged fermentation or solid state fermentation.

The cytoplasm where GA ₃ is located in the gibberella canescens is used for biosynthesis, which starts from Acetyl coenzyme A (Acetyl-CoA) synthesized by TCA cycle, synthesizes isopentenyl pyrophosphate (IPP) and isomer dimethylallyl Diphosphate (DMAPP) thereof by a mevalonate pathway, and then synthesizes precursor diyl pyrophosphate (GGPP) by using the catalysis of synthase through acyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP). Under the action of gibberellin synthesis gene cluster, two molecules of GGPP cyclize to synthesize Copperas (CPP), and kaurene is produced. Gradual oxidation by P450 monooxygenase at C-19 to give kaurenoic acid, subsequent oxidation at C-7α and C-6β to give GA ₁₂ -semialdehyde, oxidation by P450 monooxygenase at C-3β and C-7 to give GA ₁₄,GA₁₄, oxidative conversion to GA ₄, desaturation to give GA ₇, hydroxylation conversion to GA ₃.

The existing technical scheme for synthesizing GA ₃ by using gibberella canescens mainly uses mutagenesis in different modes, including chemical mutagenesis, radiation mutagenesis, ARTP mutagenesis and the like. Chinese patent (publication No. CN104892554A, CN201910304928.1 and CN 201910438160.7) respectively describe the preparation method of GA ₃, the mutagenesis result and the mutagenesis technology of ARTP, but the yield of the strain obtained by the traditional mutagenesis scheme is not improved until the yield reaches 2 g/L.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for high-yield gibberellin GA ₃ through a metabolic engineering technology, a construction method and application.

The technical scheme adopted by the invention is as follows:

The genetically engineered bacterium for producing gibberellin GA ₃ at high yield is constructed by the following method: the gibberella caner is taken as chassis fungus, and the gene coding the nitrogen source regulatory factor AreA and the gene coding the global regulatory factor Lae1 in the genome are enhanced to express, so that the genetically engineered fungus of the high-yield gibberellin GA ₃ is obtained.

Preferably, the Chaetomium is Gibberella caner (Fusarium fujikuroi) CCTCC NO: M2019378 (disclosed in prior application CN 110527630A).

The invention enhances the forward regulation and control action of the gene expressing nitrogen source regulation factor AreA and the gene expressing global regulation factor Lae1 in gibberella, so as to enhance the transcription level of the related genes (cps/ks, p450-1, p450-2, des and the like) in gibberellin anabolic pathways, and realize the purpose of accumulating gibberellin GA ₃. By combining the transformation strategies, the recombinant gibberella caner strain with high gibberellin GA ₃ yield is successfully obtained.

The invention also relates to a method for constructing the genetically engineered bacterium, which comprises the following steps: and respectively constructing in vitro assembled complete gene expression cassettes of AreA and Lae1 genes, and introducing the complete gene expression cassettes into the Chassis fungus, namely Gibberella caner, so as to obtain the genetically engineered fungus.

Preferably, the complete gene expression cassette of the AreA gene is used for linking the coding sequences of the nitrogen source regulatory factor to the gpdA promoter and trpC terminator derived from pAN7-1 plasmid.

Specifically, the coding sequence of the nitrogen source regulatory factor is shown as SEQ ID No.1, the gpdA promoter nucleotide sequence is shown as SEQ ID No.2, and the trpC terminator nucleotide sequence is shown as SEQ ID No. 3.

Preferably, the complete gene expression cassette of the Lae1 gene is used for connecting the Ptef1 promoter and Ttef terminator derived from Aspergillus nidulans to the gene sequence of the global regulatory factor.

Specifically, the gene sequence of the global regulatory factor is shown as SEQ ID No.4, the nucleotide sequence of the Ptef1 promoter is shown as SEQ ID No.5, and the nucleotide sequence of the Ttef terminator is shown as SEQ ID No. 6.

The invention also relates to application of the genetically engineered bacterium in microbial fermentation preparation of gibberellin GA ₃.

Specifically, the application is as follows: and inoculating the genetically engineered bacterium strain into a fermentation medium, performing fermentation culture at the temperature of 25-30 ℃ and the rpm of 200-300 for 150-200 hours to obtain fermentation liquor containing gibberellin GA ₃, and separating and purifying the fermentation liquor to obtain the gibberellin GA ₃.

Preferably, the fermentation medium is composed of: 70-90 g/L of corn starch, 70-90 g/L of rice flour, 10-30 g/L of soybean flour, 10-30 g/L of peanut flour, 0.5-1.5 g/L of potassium sulfate, 0.5-1.5 g/L of monopotassium phosphate, water as a solvent, natural pH and sterilization at 121 ℃ of 30 min.

Typically, the genetically engineered strain is first slant activated before fermentation, then inoculated into a seed culture medium, and cultured at 25-30 ℃ and 200-300 rpm. Then transferring the seed liquid into a fermentation medium, and culturing at the temperature of 25-30 ℃ and at the speed of 200-300 rpm. And (3) separating and purifying the culture solution to obtain gibberellin GA ₃.

The bevel activation method comprises the following steps: inoculating recombinant gibberella caner into a seed culture medium, culturing for 2 days at 25-30 ℃ and 200-300 rpm, coating on a PDA inclined plane, and culturing for 4 days at 28 ℃ to obtain a seed inclined plane.

The final concentration composition of the seed culture medium is as follows: 10-30 g/L of corn starch, 10-30 g/L of sucrose, 10-30 g/L of peanut powder, 10-30 g/L of soybean powder, 0.5-1.5 g/L of monopotassium phosphate and 0.5-1.5 g/L of magnesium sulfate, wherein the solvent is water, the pH is natural, and the sterilization is performed at 121 ℃ for 30 min.

The beneficial effects of the invention are mainly as follows: the recombinant gibberella cappa with high gibberellin GA ₃ provided by the invention has the advantages that compared with wild type, the recombinant gibberella cappa has the advantages that the overall transcription level of GA gene clusters is enhanced, the synthesis capacity of the metabolic process is improved, the transcription inhibition of GA ₃ synthesis related genes is weakened, and the carbon source substances such as starch rice flour and the nitrogen source substances such as corn flour can be better utilized for producing gibberellin GA ₃; the level of gibberellin GA ₃ produced by fermentation of the strain with the optimal performance after transformation is improved from 2 g/L to 3.25 g/L compared with that of the original strain, the production capacity of gibberellin GA ₃ is obviously improved, and the strain can be applied to large-scale gibberellin GA ₃ production.

Drawings

FIG. 1 shows the synthesis of GA ₃ in a production strain;

FIG. 2 is a flow chart of recombinant expression plasmid construction and transformation;

FIG. 3 is a graph showing the accumulation of fermentation products of an engineering strain and a starting strain;

FIG. 4 shows the fermentation results of the engineered strain after multiple passes.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

the experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the examples below, unless otherwise specified, were all conventional biochemical reagents. The term "enhancing" as used herein refers to increasing the activity of an enzyme encoded by a corresponding polynucleotide by overexpression of the gene.

Example 1: F. obtaining fujikuroi-OE AreA

The gpdA promoter fragment was amplified by PCR using primers 1 and 2 and pAN7-1 plasmid as template under the following conditions: 98 ℃ for 5min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 1 min; the temperature was continued at 72℃for 5min. The trpC terminator fragment was amplified in the same manner using primers 3 and 4 and hygromycin hyg fragment was amplified using primers 5 and 6. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragments (gpdA promoter fragment, trpC terminator fragment and hygromycin hyg1 fragment nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 7) were recovered by cutting.

Primers 7 and 8 were used by PCR, and the areA gene fragment was amplified using the genome of gibberella caner (Fusarium fujikuroi) CCTCC NO: M2019378 (CN 110527630A) as A template, and the PCR reaction conditions were as follows: 98 ℃ for 8min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 3 min; the temperature was continued at 72℃for 8min. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment (nucleotide sequence shown in SEQ ID NO. 1) was recovered by gel cutting.

The three recovered DNA fragments (gpdA promoter, areA gene, trpC terminator) were subjected to fusion PCR using primers 1 and 4 under the following conditions: 98 ℃ for 8min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 5 min; the temperature was continued at 72℃for 8min. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by gel cutting.

PUC19 vector was used as a template, and pUC19-Hyg 1-PgpdA-AreA-TttrpC vector was constructed by ligation using a one-step cloning kit ClonExpress II One Step Cloning Kit (purchased from Nanjinopran Vazyme), and the ligation product was transformed into E. Coli DH 5. Alpha. Receptor bacteria, which were spread on LB solid plates containing 100 mg/L ampicillin resistance, and incubated at 37℃for 12h. Single colonies are randomly picked into LB liquid medium with 100 mg/L of ampicillin resistance, cultured for 12 hours at 37 ℃, bacterial cells are collected, and plasmids are extracted to obtain pUC19-Hyg1-PgpdA-AreA-TtrPC vectors.

The preparation of the gibberella canescens protoplast, the transformation of exogenous fragments, the resuscitating culture scheme and the verification scheme are described in CN 113832041A. The transformed single colonies were subjected to PCR with primers 1 and 4 to confirm integration of the exogenous fragment into the genome. The strain thus constructed was designated Fusarium fujikuroi-OE:AreA. The plasmid construction and transformation procedure is shown in FIG. 2.

Table 1: primer sequences

Example 2: f. Fujikuroi-OE Lae1 acquisition

The tef1 promoter fragment was amplified by PCR using primers 9 and 10 using the A.nidulans genome as a template, and the PCR reaction conditions were as follows: 98 ℃ for 5min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 1 min; the temperature was continued at 72℃for 5min. The tef1 terminator fragment was amplified in the same manner using primers 11 and 12, and hygromycin hyg2 fragment was amplified using pAN7-1 plasmid as a template and primers 13 and 14. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragments (tef 1 promoter fragment, tef1 terminator fragment and hygromycin hyg2 fragment nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 8) were recovered by cutting.

Primers 15 and 16 are used for PCR, and the genome of the gibberella caner (Fusarium fujikuroi) CCTCC NO: M2019378 is used as a template for amplification to obtain a lae1 gene fragment, wherein the PCR reaction conditions are as follows: 98 ℃ for 8min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 1 min; the temperature was continued at 72℃for 8min. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment (nucleotide sequence shown in SEQ ID NO. 4) was recovered by gel cutting.

The three recovered DNA fragments (tef 1 promoter, lae1 gene, tef2 terminator) were subjected to fusion PCR using primers 9 and 12 under the following conditions: 98 ℃ for 8min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 3 min; the temperature was continued at 72℃for 8min. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by gel cutting.

PUC19 vector was used as a template, and pUC19-Hyg2-Ptef1-Lae1-Ttef1 vector was constructed by ligation using a one-step cloning kit ClonExpress II One Step Cloning Kit (purchased from Nanjinopran Vazyme), and the ligation product was transformed into E.coli DH 5. Alpha. Receptor bacteria, spread on LB solid plates containing 100 mg/L ampicillin resistance, and cultured at 37℃for 12 hours. Single colonies are randomly picked into LB liquid medium with 100 mg/L of ampicillin resistance, cultured for 12 hours at 37 ℃, thalli are collected, and plasmids are extracted to obtain pUC19-Hyg2-Ptef1-Lae1-Ttef vectors.

The single colony of gibberella canescens after transformation of the vector was subjected to PCR with primers 9 and 12 for confirming integration of the exogenous fragment into the genome. The strain thus constructed was designated Fusarium fujikuroi-OE Lae1.

Table 2: primer sequences

Example 3: f. Fujikuroi-OE AreA OE Lae1 acquisition

The complete Lae1 expression cassette was amplified by PCR using primers 17 and 18, using pUC19-Hyg2-Ptef1-Lae1-Ttef1 vector as template in example 2. The PCR conditions were as follows: 98 ℃ for 8min; repeating 30 cycles at 98 deg.c for 30s,60 deg.c for 30s and 72 deg.c for 4 min; the temperature was continued at 72℃for 8min. The PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by gel cutting.

PUC19-Hyg1-PgpdA-AreA-TtrPC vector is used as a template, and is connected through a one-step cloning kit ClonExpress II One Step Cloning Kit (purchased from Nanjuzhan vazyme), so that pUC19-Hyg1-PgpdA-AreA-TtrPC-Ptef1-Lae1-Ttef1 vector is constructed, and the connection product is transformed into E.coli DH5 alpha receptor bacteria, coated on LB solid plates with 100 mg/L ampicillin resistance, and cultured for 12 hours at 37 ℃. Single colonies are randomly picked into LB liquid medium with 100 mg/L of ampicillin resistance, cultured for 12 hours at 37 ℃, thalli are collected and plasmids are extracted to obtain pUC19-Hyg1-PgpdA-AreA-TtrpC-Ptef1-Lae1-Ttef1 vectors.

The single colony of gibberella canescens after transformation of the vector was subjected to PCR with primers 19 and 20 for confirming integration of the exogenous fragment into the genome. The strain thus constructed was designated Fusarium fujikuroi-OE AreA OE Lae1.

Table 3: primer sequences

Example 4: shake flask fermentation verification of gibberellin-producing GA ₃ engineering bacteria of different genotypes

The modified strains and the starting strains constructed in examples 1,2 and 3 were inoculated into 25 mL seed medium, respectively, and cultured at 28℃and 250 rpm to be used as precultures. 48 After h, 2.8 mL precultures were inoculated into 500mL shake flasks containing 40 mL fermentation medium and then incubated at 28℃for 7 days at 250 rpm. The GA ₃ content was measured according to the HPLC detection method of example 7 of patent CN 113832041A. The change of GA ₃ content in the fermentation broth during fermentation is shown in figure 3.

The seed culture medium is prepared according to the following method: corn starch 20 g/L, sucrose 20 g/L, peanut powder 20 g/L, soybean powder 20 g/L, monopotassium phosphate 1.0g/L, magnesium sulfate 1.0g/L, natural pH, and sterilization at 121deg.C 30 min.

Fermentation medium composition: 80g/L of corn starch, 80g/L of rice flour, 20 g/L of soybean flour, 20 g/L of peanut flour, 1.0 g/L of potassium sulfate, 1.0 g/L of monopotassium phosphate, tap water as a solvent, natural pH and sterilization at 121 ℃ of 30 min.

The fermentation yields of the strains of different genotypes are shown in Table 4.

TABLE 4 Table 4

Example 5: passage verification of high-yield gibberellin GA ₃ engineering bacteria

According to the composition and culture conditions of the slant culture medium of patent CN113832041A, the modified strains constructed in examples 1,2 and 3 are selected, and after continuous 5 passages, the yield of gibberellin GA ₃ is stable and is improved by more than 60% compared with the original strain through the same culture method and HPLC analysis as in example 4. The passage verification result is shown in figure 4, which shows that the gibberellin-producing GA ₃ engineering bacteria constructed in the invention has passage stability.

Sequence listing

<110> Zhejiang university of industry

Gene engineering bacteria for producing gibberellin GA 3 with high yield of <120>, construction method and application

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ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc tcaggagctg gccagcggcg 1920

cagaccggga acacaagctg gcagtcgacc catccggtgc tctgcactcg acctgctgag 1980

gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc gcccggtgtg tcggcggggt 2040

tgacaaggtc gttgcgtcag tccaacattt gttgccatat tttcctgctc tccccaccag 2100

ctgctctttt cttttctctt tcttttccca tcttcagtat attcatcttc ccatccaaga 2160

acctttattt cccctaagta agtactttgc tacatccata ctccatcctt cccatccctt 2220

attcctttga acctttcagt tcgagctttc ccacttcatc gcagcttgac taacagctac 2280

cccgcttgag cagacatcac c 2301

<210> 3

<211> 770

<212> DNA

<213> Unknown (Unknown)

<400> 3

ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt 60

tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg 120

ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag 180

aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg 240

cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat agcttagcag 300

agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg 360

agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg 420

acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac cagcncctgg 480

gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc 540

atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata gtaaccatgc 600

atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact 660

ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt 720

ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 770

<210> 4

<211> 1261

<212> DNA

<213> Unknown (Unknown)

<400> 4

atggttgtaa tgcctcctca aaacaggtat gctatagatc tctttgtcgg gcagccaatc 60

ttacatatgc aatgcagcgt gaacgagtcg gaagggcgat accttcaaga tgggttctgg 120

caacatggcc gtttctatgg ctcttggaaa cctggcaaat acttatttcc aattgactca 180

gtaagtcgtc tgaaaatgac aatatcgtac gccacctgac cagcaacagg aagagttgaa 240

tcgtttggat atatttcata aagtcttcct tctggcacga gacaataagc catttctagc 300

tcccatcaga cgtacctcgc ccagaatcat ggatattggt acgggcacag gtatctgggc 360

aatcaatgtg gccgaagagt atgaatgcct gtgccattga tctcgtttat caccacgtta 420

ctgacctaaa gaagatgtct ctcagatgct cagatcatgg ccgtagacct gaatcagatt 480

caaccagcat tgtatgctcg ctcacactcg ctctttgtag gccatcggct aatgtagata 540

ggatcccccc tggctttatg ccaaagcagt atgacattga agaaccgtct tgggggcccc 600

tactggcgga ctgcgatttg attcatatgc gaatgctact tggcagtatt cagacggact 660

tgtggcctca agtctaccac aatgcctttg agtatgttcg gaactatatc tttgagcatc 720

acactgatac ctcactaggc acttgactcc aggcatcggc ttcctggagc acattgaggt 780

tgactggata ccccgatgtg atgatgatga acgcccagca aattccgcat tcgtgaagtg 840

ggcagagctt tttctcgacg gcatggatcg attcaaccgc agtgtcagag tcataccgca 900

ggaacatcgg caaatgctcg aagctacagg tttcacagat gtcaagcagg aggtgatcaa 960

ggcctatgtt tgcccctggt ctgctgatcg aaatgaacgt gaaatcgctc gatggttcaa 1020

catcggactg tctcatagtc tggaggccat gagcttaaaa cccctaatcg agaaactcgg 1080

attcgaggcc gaggatgtcc gtgagctatg tgagagagcg aagcgcgaaa cgtgtgtttt 1140

gcgctatcac acttattgta atatgtgagt ctacaaacgc ctcctaccga cacacttgat 1200

ggcatattaa cgctttcgca gtcatgtctg gacggctagg aagcccggac ctcaacagta 1260

a 1261

<210> 5

<211> 886

<212> DNA

<213> Unknown (Unknown)

<400> 5

cgagacagca gaatcaccgc ccaagttaag cctttgtgct gatcatgctc tcgaacgggc 60

caagttcggg aaaagcaaag gagcgtttag tgaggggcaa tttgactcac ctcccaggca 120

acagatgagg ggggcaaaaa gaaagaaatt ttcgtgagtc aatatggatt ccgagcatca 180

ttttcttgcg gtctatcttg ctacgtatgt tgatcttgac gctgtggatc aagcaacgcc 240

actcgctcgc tccatcgcag gctggtcgca gacaaattaa aaggcggcaa actcgtacag 300

ccgcggggtt gtccgctgca aagtacagag tgataaaagc cgccatgcga ccatcaacgc 360

gttgatgccc agctttttcg atccgagaat ccaccgtaga ggcgatagca agtaaagaaa 420

agctaaacaa aaaaaaattt ctgcccctaa gccatgaaaa cgagatgggg tggagcagaa 480

ccaaggaaag agtcgcgctg ggctgccgtt ccggaaggtg ttgtaaaggc tcgacgccca 540

aggtgggagt ctaggagaag aatttgcatc gggagtgggg cgggttaccc ctccatatcc 600

aatgacagat atctaccagc caagggtttg agcccgcccg cttagtcgtc gtcctcgctt 660

gcccctccat aaaaggattt cccctccccc tcccacaaaa ttttctttcc cttcctctcc 720

ttgtccgctt cagtacgtat atcttccctt ccctcgcttc tctcctccat ccttctttca 780

tccatctcct gctaacttct ctgctcagca cctctacgca ttactagccg tagtatctga 840

gcacttctcc cttttatatt ccacaaaaca taacacaacc ttcacc 886

<210> 6

<211> 489

<212> DNA

<213> Unknown (Unknown)

<400> 6

gcggacattc gatttatgcc gttatgactt ccttaaaaaa gcctttacga atgaaagaaa 60

tggaattaga cttgttatgt agttgattct acaatggatt atgattcctg aacttcaaat 120

ccgctgttca ttattaatct cagctcttcc cgtaaagcca atgttgaaac tattcgtaaa 180

tgtacctcgt tttgcgtgta ccttgcttat cacgtgatat tacatgacct ggacagagtt 240

ctgcgcgaaa gtcataacgt aaatcccggg cggtaggtgc gtcccgggcg gaaggtagtt 300

ttctcgtcca ccccaacgcg tttatcaacc tcaactttca acaaccatca tgccaccaaa 360

agcgcgtaaa acaaagcgag atttgattga gcaagagggc aggatccaat gcgcgattca 420

agacattaaa aatggaaaat ttcaaaaaat tgcgcccgca gcgcgtgcat acaaaattca 480

tcccaatac 489

<210> 7

<211> 1058

<212> DNA

<213> Unknown (Unknown)

<400> 7

cactccacat ctccactcga atgcctgaac tcaccgcgac gtctgtcgag aagtttctga 60

tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 120

ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 180

gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 240

aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac 300

agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 360

cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 420

tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 480

atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 540

aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 600

acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 660

actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 720

ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 780

ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 840

agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 900

tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 960

tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 1020

gtccgagggc aaaggaatag ggtacctcta gaaagctt 1058

<210> 8

<211> 1058

<212> DNA

<213> Unknown (Unknown)

<400> 8

tacaaaattc atcccaatac atgcctgaac tcaccgcgac gtctgtcgag aagtttctga 60

tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg 120

ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg 180

gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg 240

aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac 300

agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg 360

cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat 420

tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg 480

atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc 540

aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc 600

acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg 660

actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga 720

ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc 780

ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc 840

agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa 900

tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg 960

tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc 1020

gtccgagggc aaaggaatag ggtacctcta gaaagctt 1058

Claims

1. The genetically engineered bacterium for producing gibberellin GA ₃ at high yield is constructed by the following method: the gibberella caner is taken as chassis bacteria, the gene encoding nitrogen source regulatory factor AreA and the gene encoding global regulatory factor Lae1 in the genome are enhanced to express, and the genetically engineered bacteria of the high-yield gibberellin GA ₃ are obtained, and the chassis bacteria are the gibberella caner (Fusarium fujikuroi) CCTCC NO: M2019378;

The method comprises the following steps: respectively constructing in vitro assembled complete gene expression cassettes of AreA and Lae1 genes, and introducing the complete gene expression cassettes into the Chassis fungus which is Alternaria fumartensii to obtain the genetically engineered bacteria;

The complete gene expression cassette of the AreA gene is used for connecting a gpdA promoter and a trpC terminator which are derived from pAN7-1 plasmid with a coding sequence of a nitrogen source regulatory factor, wherein the coding sequence of the nitrogen source regulatory factor is shown as SEQ ID No.1, the nucleotide sequence of the gpdA promoter is shown as SEQ ID No.2, and the nucleotide sequence of the trpC terminator is shown as SEQ ID No. 3;

the complete gene expression cassette of the Lae1 gene is used for connecting a Ptef1 promoter and Ttef terminator derived from Aspergillus nidulans with a gene sequence of a global regulatory factor, wherein the gene sequence of the global regulatory factor is shown as SEQ ID No.4, the nucleotide sequence of the Ptef1 promoter is shown as SEQ ID No.5, and the nucleotide sequence of the Ttef terminator is shown as SEQ ID No. 6.

2. The use of the genetically engineered bacterium of claim 1 in the preparation of gibberellin GA ₃ by microbial fermentation.

3. The application according to claim 2, characterized in that it is: and inoculating the genetically engineered bacterium strain into a fermentation medium, performing fermentation culture at the temperature of 25-30 ℃ and the rpm of 200-300 for 150-200 hours to obtain fermentation liquor containing gibberellin GA ₃, and separating and purifying the fermentation liquor to obtain the gibberellin GA ₃.

4. Use according to claim 3, characterized in that the fermentation medium consists of: 70-90 g/L of corn starch, 70-90 g/L of rice flour, 10-30 g/L of soybean flour, 10-30 g/L of peanut flour, 0.5-1.5 g/L of potassium sulfate, 0.5-1.5 g/L of monopotassium phosphate, water as a solvent, natural pH and sterilization at 121 ℃ of 30 min.