CN114517161A - High yield gibberellin GA3Genetically engineered bacterium, construction method and application - Google Patents

Abstract

The invention relates to a high-yield gibberellin GA₃The gene engineering bacteria, the construction method and the application. The invention enhances the positive regulation effect of the expression nitrogen source regulation factor AreA gene and the global regulation factor Lae1 gene in the gibberellin anabolism path by enhancing the expression of the nitrogen source regulation factor AreA gene and the global regulation factor Lae1 gene in the gibberellin anabolism path, improves the transcription level of the related genes, and improves the gibberellin GA₃Accumulation of (2). The high-yield gibberellin GA provided by the invention₃Compared with wild type, the reconstructed Gibberella fujikuroi enhances the whole transcription level of GA gene cluster, improves the synthesis capacity in the metabolic process, weakens the GA₃Transformation of synthetic related genesThe gibberellin GA can be better performed by utilizing carbon source substances and nitrogen source substances through transcription inhibition₃The strain with the best performance after being modified is fermented to produce the gibberellin GA₃Compared with the original strain, the level of the gibberellin is improved from 2 g/L to 3.25 g/L, and the gibberellin GA is obviously improved₃Production capacity, applicable to large-scale gibberellin GA₃In the production process.

Description

High yield gibberellin GA3Genetically engineered bacterium, construction method and application

Technical Field

The invention relates to a high-yield gibberellin GA ₃The gene engineering bacteria, the construction method and the application.

Background

Gibberellin (Gibberellin Acid, GA)₃) Is a plant growth hormone synthesized by plants and partial fungi. Due to its special ability to stimulate plant growth, it is widely used in agriculture, horticulture and wine production. The discovery of Gibberella fujikuroi by Japanese plant pathologist in the early stage of research on rice bakanae disease, and subsequent research proves that Gibberella fujikuroi naturally has complete GA₃And (4) synthetic ability. GA₃Is a tetracyclic diterpene carboxylic acid with a molecular formula C₁₉H₂₂O₆The molecular weight is 346.37, the melting point is 233-235 ℃, the buffer solution is easily soluble in alcohol, acetone, ethyl acetate, sodium bicarbonate solution and phosphate buffer solution with the pH value of 6.2, and the buffer solution is hardly soluble in water and ether. GA₃The secondary metabolite is generally obtained by liquid fermentation, solid fermentation and plant extraction. Current GA₃The production of (A) mainly depends on liquid submerged fermentation or solid state fermentation.

GA in Gibberella fujikuroi₃The synthesis site of (2) is located in the cytoplasm, and biosynthesis of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl Diphosphate (DMAPP) is carried out from Acetyl coenzyme A (Acetyl-CoA) cyclically synthesized by TCA via the mevalonate pathway, and then precursor diphosphoric acid (GGPP) is synthesized from farnesyl pyrophosphate (FPP) via pyrophosphoric acid (GPP) by synthetase. Under the action of gibberellin synthesis gene cluster, two molecules of GGPP cyclize to synthesize copaiba pyrophosphate (CPP), and then kaurene is produced. Step-wise oxidation by P450 monooxygenase at C-19 to yield kaurenoic acid, followed by oxidation at the C-7 alpha and C-6 beta positions to yield GA ₁₂Oxidation of semialdehyde, P450 monooxygenase at C-3. beta. and C-7 to GA₁₄，GA₁₄Conversion to GA by oxidation₄Production of GA by desaturation₇Is converted into GA by hydroxylation₃。

Existing GA synthesis method for Gibberella fujikuroi₃The technical scheme of (1) mainly uses different partiesThe mutagenesis of formula (II) is mainly chemical mutagenesis, radiation mutagenesis, ARTP mutagenesis and the like. Chinese invention patents (publication Nos. CN104892554A, CN201910304928.1 and CN 201910438160.7) respectively describe GA₃The preparation method, the mutagenesis result and the mutagenesis technology of ARTP, but the strain yield obtained by the traditional mutagenesis scheme is difficult to be improved after reaching 2 g/L.

Disclosure of Invention

The invention aims to provide high-yield gibberellin GA by a metabolic engineering technology₃The gene engineering bacteria, the construction method and the application.

The technical scheme adopted by the invention is as follows:

high yield gibberellin GA₃The genetically engineered bacterium is constructed and obtained by the following method: taking the red vine mold as the Chassis bacteria, enhancing the expression of the gene of the coding nitrogen source regulatory factor AreA and the gene of the coding global regulatory factor Lae1 in the genome of the red vine mold, and obtaining the high-yield gibberellin GA₃The genetically engineered bacterium of (1).

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

The invention enhances the positive regulation and control effect on related genes (cps/ks, p450-1, p450-2, des and the like) in the gibberellin anabolism path by enhancing the gene for expressing nitrogen source regulation factor AreA and the gene for expressing global regulation and control factor Lae1 in the gibberella lutea, improves the transcription level of the related genes, and realizes the accumulation of gibberellin GA₃The object of (1). By combining the transformation strategies, the invention successfully obtains the high-yield gibberellin GA₃The recombinant strain of gibberella gambieri of (1).

The invention also relates to a method for constructing the genetic engineering bacteria, which comprises the following steps: in-vitro assembled complete gene expression cassettes of the AreA and Lae1 genes are respectively constructed and are introduced into a chassis bacterium, namely, the Gibberella fujikuroi, so as to obtain the genetic engineering bacterium.

Preferably, the complete gene expression cassette of the AreA gene is used from the pAN7-1 plasmidgpdAPromoters andtrpCcoding sequence of terminator connected nitrogen source regulatory factor。

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

Preferably, the complete gene expression cassette of the Lae1 gene is connected with the gene sequence of a global regulatory factor by using a Ptef1 promoter and a Tref 1 terminator which are derived from aspergillus nidulans.

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 Tref 1 terminator is shown as SEQ ID No. 6.

The invention also relates to the preparation of gibberellin GA by the genetic engineering bacteria in microbial fermentation₃The use of (1).

Specifically, the application is as follows: inoculating the genetic engineering strain into a fermentation culture medium, and performing fermentation culture for 150-200 h at 25-30 ℃ and 200-300 rpm to obtain GA containing gibberellin₃The fermentation liquor of (1) is separated and purified to obtain the gibberellin GA₃。

Preferably, 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 powder, 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 ℃ for 30 min.

Generally, before the genetic engineering strain is fermented, slant activation is firstly carried out, and then the genetic engineering strain is inoculated to a seed culture medium and cultured under the conditions of 25-30 ℃ and 200-300 rpm. Then transferring the seed liquid into a fermentation culture medium, and culturing at 25-30 ℃ and 200-300 rpm. Separating and purifying the culture solution to obtain gibberellin GA ₃。

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

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

The invention has the following beneficial effects: the high-yield gibberellin GA provided by the invention₃Compared with wild type, the reconstructed Gibberella fujikuroi enhances the whole transcription level of GA gene cluster, improves the synthesis capacity in the metabolic process, weakens the GA₃The transcription inhibition of the synthesis related gene can better utilize carbon source substances such as starch rice flour and the like and nitrogen source substances such as peanut flour corn flour and the like to carry out gibberellin GA₃The production of (1); fermentation of improved strain with optimal performance for producing gibberellin GA₃Compared with the original strain, the level of the gibberellin is improved from 2 g/L to 3.25 g/L, and the gibberellin GA is obviously improved₃Production capacity, applicable to large-scale gibberellin GA ₃In the production process.

Drawings

FIG. 1 is GA in the production strains₃The synthesis process of (2);

FIG. 2 is a diagram showing the construction process and transformation process of recombinant expression plasmid;

FIG. 3 is a curve of fermentation product accumulation for an engineering strain and a starting strain;

FIG. 4 shows the fermentation results of the engineering strain after multiple rounds of passage.

Detailed Description

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

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

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

Example 1:F. fujikuroiobtaining of OE AreA

Obtained by PCR using primers 1 and 2 and pAN7-1 plasmid as a templategpdAPromoter fragment, PCR reaction stripThe following parts: 5min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; the temperature of 72 ℃ is continued for 5 min. Amplified in the same manner using primers 3 and 4trpCTerminator fragment, amplified using primers 5 and 6 to obtain hygromycinhygAnd (3) fragment. PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by cutting the gel ( gpdAA promoter fragment,trpCTerminator fragment and hygromycinhyg1The nucleotide sequences of the fragments are respectively shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 7).

By PCR using primers 7 and 8, and Bacillus cereus (A. lutescens, B. lutescens, C. lutescens, et al), (B. lutescens, et al)Fusarium fujikuroi) M2019378 (CN 110527630A) genome as template and obtained by amplificationareAThe gene fragment and the PCR reaction conditions are as follows: 8min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 3 min; the temperature of 72 ℃ is continued for 8 min. 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 cutting the gel.

Three DNA fragments recovered (gpdAA promoter,areAthe gene(s) is (are),trpCterminator) fusion PCR was performed using primers 1 and 4, with the PCR reaction conditions as follows: 8min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 5 min; the temperature of 72 ℃ is continued for 8 min. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by cutting the gel.

Using pUC19 vector as a template, the fragment was ligated with the One-Step Cloning Kit Cloneexpress II One Step Cloning Kit (purchased from Vazyme of Kinzoka, Nanjing) to construct pUC19-Hyg1-PgpdA-AreA-TtrpCVector, ligation product transformation toE. coliThe DH 5. alpha. recipient strain was plated on LB solid plates containing 100 mg/L of ampicillin resistance at the final concentration and cultured at 37 ℃ for 12 hours. Randomly picking a single colony to LB liquid culture medium containing 100 mg/L ampicillin resistance at the final concentration, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain pUC19-Hyg1- PgpdA-AreA-TtrpCAnd (3) a carrier.

Preparation of a gibberella granatum protoplast, exogenous fragment transformation, a recovery culture scheme and a verification scheme as described in CN 113832041A. The transformed single colonies were subjected to PCR with primers 1 and 4 to confirm the integration of the foreign fragment into the genome. The strain thus constructed was named Fusarium fujikuroi-OE AreA. The plasmid construction and transformation process is shown in FIG. 2.

Table 1: primer sequences

Example 2:F.fujikuroiobtaining of-OE: Lae1

Obtained by PCR using primers 9 and 10 and using the Aspergillus nidulans genome as a templatetef1Promoter fragment, PCR reaction conditions as follows: 5min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; the temperature of 72 ℃ is continued for 5 min. Amplified in the same manner using primers 11 and 12tef1The terminator fragment was amplified using pAN7-1 plasmid as template and primers 13 and 14 to obtain hygromycinhyg2And (3) fragment. PCR products were detected by 1.0% agarose gel electrophoresis and the purified fragments were recovered by cutting the gel (tef1A promoter fragment,tef1Terminator fragment and hygromycinhyg2The nucleotide sequences of the fragments are respectively shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 8).

Obtained by PCR using primers 15 and 16 and M2019378 genome as template for amplification lae1The gene fragment and the PCR reaction conditions are as follows: 8min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 1 min; the temperature of 72 ℃ is continued for 8 min. 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 cutting.

Three DNA fragments recovered (tef1A promoter,lae1the gene(s) is (are),tef2terminator) fusion PCR was performed using primers 9 and 12, and PCR reaction conditions were as follows: 8min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 3 min; the temperature of 72 ℃ is continued for 8 min. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by cutting the gel.

Using pUC19 vector as a template, the fragment was ligated with the One-Step Cloning Kit Cloneexpress II One Step Cloning Kit (purchased from Vazyme of Kinzoka, Nanjing) to construct pUC19-Hyg2-Ptef1-Lae1-Ttef1Vector, ligation product transformation toE. coliThe DH 5. alpha. recipient strain was plated on LB solid plates containing 100 mg/L of ampicillin resistance at the final concentration and cultured at 37 ℃ for 12 hours. Randomly picking a single colony to LB liquid culture medium containing 100 mg/L ampicillin resistance at the final concentration, culturing for 12h at 37 ℃, collecting thalli and extracting plasmids to obtain pUC19-Hyg2-Ptef1-Lae1-Ttef1And (3) a carrier.

The single colony of the transformed vector of the red-eared fusarium was subjected to PCR using primers 9 and 12 to confirm that the foreign fragment was integrated into the genome. The strain thus constructed was named Fusarium fujikuroi-OE: Lae 1.

Table 2: primer sequences

Example 3:F.fujikuroiobtaining of-OE: AreA OE: Lae1

Using primers 17 and 18 by PCR, pUC19-Hyg2-Ptef1-Lae1-Ttef1The vector is used as a template, and the complete expression cassette of Lae1 is amplified. The PCR reaction conditions were as follows: 8min at 98 ℃; 30 cycles of 98 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 4 min; the temperature of 72 ℃ is continued for 8 min. The PCR product was detected by 1.0% agarose gel electrophoresis and the purified fragment was recovered by cutting the gel.

By pUC19-Hyg1-PgpdA-AreA-TtrpCVector as template, was ligated by One-Step Cloning Kit Clonexpress II One Step Cloning Kit (purchased from Vazyme of Kinza of Nanjing) to construct pUC19-Hyg1-PgpdA-AreA-TtrpC-Ptef1-Lae1-Ttef1The vector and the ligation product were transformed into E.coli DH 5. alpha. recipient strain, plated on LB solid plates containing 100 mg/L final ampicillin resistance, and cultured at 37 ℃ for 12 hours. Randomly picking single colony to LB liquid culture medium containing 100 mg/L ampicillin resistance at final concentration, culturing at 37 deg.C for 12h, collecting thallus and extracting plasmid to obtain pUC19-Hyg1-PgpdA-AreA-TtrpC-Ptef1-Lae1-Ttef1And (3) a carrier.

The vector-transformed single colonies of Gibberella fujikuroi were subjected to PCR using primers 19 and 20 to confirm the integration of the foreign fragment into the genome. The strain thus constructed was named Fusarium fujikuroi-OE AreA OE Lae 1.

Table 3: primer sequences

Example 4: different genotypes produce gibberellin GA₃Shake flask fermentation verification of engineering bacteria

The engineered strain and the starting strain constructed in examples 1, 2 and 3 were inoculated in 25 mL of seed medium, respectively, and cultured at 28 ℃ and 250 rpm as a preculture. After 48 h, 2.8 mL of preculture were inoculated into a 500 mL shake flask containing 40 mL of fermentation medium and then incubated at 28 ℃ and 250 rpm for 7 days. GA detection according to the HPLC detection method of example 7 of patent CN113832041A₃And (4) content. During the fermentation process, GA in the fermentation liquor₃The content changes are shown in FIG. 3.

The seed culture medium is prepared by the following method: 20 g/L of corn starch, 20 g/L of cane sugar, 20 g/L of peanut powder, 20 g/L of soybean powder, 1.0g/L of monopotassium phosphate and 1.0g/L of magnesium sulfate, natural pH and sterilization at 121 ℃ for 30 min.

The fermentation medium comprises the following components: 80g/L of corn starch, 80g/L of rice flour, 20 g/L of soybean flour, 20 g/L of peanut powder, 1.0g/L of potassium sulfate, 1.0g/L of monopotassium phosphate and tap water as a solvent, wherein the pH is natural, and the sterilization is carried out at 121 ℃ for 30 min.

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

TABLE 4

Example 5: high yield gibberellin GA₃Passage verification of engineering bacteria

According to the slant culture medium composition and culture conditions of patent CN113832041A, the modified strains constructed in examples 1, 2 and 3 were selected, passed through 5 serial passages, and subjected to the same culture method and HPLC analysis as in example 4 to obtain gibberellin GA ₃The yield is stable and is improved by more than 60 percent compared with the original strain. Passage verificationThe results are shown in FIG. 4, which illustrates the gibberellin-producing GA constructed in the present invention₃The engineering bacteria have passage stability.

Sequence listing

<110> Zhejiang industrial university

<120>High yield gibberellin GA₃The gene engineering bacterium, the construction method and the application

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gccagacagc tctggcggct ctgaggtgca gtggatgatt attaatccgg gaccggccgc 1560

ccctccgccc cgaagtggaa aggctggtgt gcccctcgtt gaccaagaat ctattgcatc 1620

atcggagaat atggagcttc atcgaatcac cggcagtaag cgaaggagaa tgtgaagcca 1680

ggggtgtata gccgtcggcg aaatagcatg ccattaacct aggtacagaa gtccaattgc 1740

ttccgatctg gtaaaagatt cacgagatag taccttctcc gaagtaggta gagcgagtac 1800

ccggcgcgta agctccctaa ttggcccatc cggcatctgt agggcgtcca aatatcgtgc 1860

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 (10)

1. High yield gibberellin GA₃The genetically engineered bacterium is constructed and obtained by the following method: taking the red vine mold as the Chassis bacteria, enhancing the expression of the gene of the coding nitrogen source regulatory factor AreA and the gene of the coding global regulatory factor Lae1 in the genome of the red vine mold, and obtaining the high-yield gibberellin GA₃The genetically engineered bacterium of (1).

2. The genetically engineered bacterium of claim 1, wherein the basidiomycete is gibberella gambieri (Chi) and (C)Fusarium fujikuroi）CCTCC NO: M 2019378。

3. A method for constructing the genetically engineered bacterium of claim 1, the method comprising: in-vitro assembled complete gene expression cassettes of the AreA and Lae1 genes are respectively constructed and are introduced into a chassis bacterium, namely, the Gibberella fujikuroi, so as to obtain the genetic engineering bacterium.

4. The method of claim 3, wherein: the complete gene expression cassette of the AreA gene, which was derived from the pAN7-1 plasmid gpdAPromoters andtrpCthe terminator is connected with the coding sequence of the nitrogen source regulatory factor.

5. The method of claim 3, wherein: the coding sequence of the nitrogen source regulatory factor is shown as SEQ ID No.1, and the nitrogen source regulatory factor is expressed in sequencegpdAThe nucleotide sequence of the promoter is shown as SEQ ID No.2trpCThe nucleotide sequence of the terminator is shown as SEQ ID No.3。

6. The method of claim 3, wherein: the complete gene expression cassette of the Lae1 gene is used for connecting a Ptef1 promoter and a Tref 1 terminator from aspergillus nidulans with a gene sequence of a global regulatory factor.

7. The method of claim 3, 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 Tref 1 terminator is shown as SEQ ID No. 6.

8. Preparation of gibberellin GA by microbial fermentation of the genetically engineered bacteria of claim 1₃The use of (1).

9. The use according to claim 6, characterized in that the use is: inoculating the genetic engineering strain into a fermentation culture medium, and performing fermentation culture for 150-200 h at 25-30 ℃ and 200-300 rpm to obtain GA containing gibberellin₃The fermentation liquor of (1) is separated and purified to obtain the gibberellin GA ₃。

10. Use according to claim 7, 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 powder, 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 ℃ for 30 min.

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