CN115181754B - Pseudomonas aeruginosa genetically engineered bacterium for high-yield phenazine-1-carboxylic acid and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a pseudomonas aeruginosa genetically engineered bacterium for high-yield phenazine-1-carboxylic acid, a construction method and application thereof. The Pseudomonas aeruginosa genetic engineering bacteria for high-yield phenazine-1-carboxylic acid is obtained by knocking out phzO gene, degU gene, tctB gene and hppA gene in the genome of Pseudomonas aeruginosa (Pseudomonas chlororaphis) Qlu-1, wherein the Pseudomonas aeruginosa Qlu-1 is preserved in China center for type culture collection, the preservation address is China, the university of Wuhan and the university of Wuhan, the preservation date is 05 month 08 in 2020, and the preservation number is CCTCC NO: m2020108. The 48h phenazine-1-carboxylic acid fermentation yield of the strain reaches 4787.2mg/L, the productivity of the strain is greatly improved, and a solid foundation is provided for the industrialization of subsequent engineering strains.

Description

Pseudomonas aeruginosa genetically engineered bacterium for high-yield phenazine-1-carboxylic acid and construction method and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a pseudomonas aeruginosa genetically engineered bacterium for high-yield phenazine-1-carboxylic acid, a construction method and application thereof.

Background

The use of pesticides has long been an important means of increasing both production and income in agriculture. Most of the pesticides on the market at present are chemical pesticides produced through harsh chemical synthesis. Chemical pesticides are not ignored in the aspect of crop yield maintenance and yield increase, however, toxic and harmful substances are often generated in the production process, and the pesticides used in farmlands are also often not easy to degrade, so that serious pesticide residues are caused, and ecological environment pollution is caused. Therefore, searching for alternatives to chemical pesticides is an important research content of current technological workers. The biological pesticide is a biological active substance synthesized by microorganisms and other organisms naturally, has good control effect on plant diseases and insect pests of crops, and has the characteristics of easy biodegradation and the like. Therefore, the biological pesticide becomes a good substitute for chemical pesticides.

Phenazine-1 carboxylic acid (PCA) is a biologically active substance, which was granted to agricultural certificates by the Ministry of the people's republic of China in 2011 because of its good effect on crop diseases and insect pests, and formally named "shenzinomycin", the production of phenazine-1 carboxylic acid has been changed from chemical production to fermentation production by Pseudomonas aeruginosa. However, pseudomonas aeruginosa is a relatively common opportunistic pathogen, and is not suitable for popularization and application as a production strain in the long term. Compared with pseudomonas aeruginosa, pseudomonas aeruginosa also contains a core gene component phzABCDEFG synthesized by phenazine matters, but is not a conditional pathogen and can be used as an alternative strain for producing PCA.

Pseudomonas aeruginosa Qlu-1 is a Pseudomonas aeruginosa screened from the root of vegetable green house peppers in a garden, and the modified gene phzO containing phzABCDEFG and phenazine is obtained by sequencing, so that phenazine-1-carboxylic acid and 2-hydroxy phenazine can be produced. Studies have shown that phzO enzymes catalyze the conversion of phenazine-1-carboxylic acid to 2-hydroxyphenazine, and thus knocking out phzO can cause strains to accumulate phenazine-1-carboxylic acid. The phzO gene of pseudomonas aeruginosa Qlu-1 is knocked out and fermented, and the highest yield of PCA is about 408mg/L, which can not reach the popularization and application level.

Based on the method, the yield of the engineering strain is improved by a molecular biology method, and the preparation is made for popularization and application.

Disclosure of Invention

Aiming at the technical problem that the PCA yield is still low after the phzO gene of the pseudomonas aeruginosa Qlu-1 is knocked out, the invention provides the pseudomonas aeruginosa genetic engineering bacterium for high-yield phenazine-1-carboxylic acid, and the construction method and the application thereof, the 48h phenazine-1-carboxylic acid fermentation yield of the strain reaches 4787.2mg/L, the productivity of the strain is greatly improved, and a solid foundation is provided for the industrialization of subsequent engineering strains.

In a first aspect, the invention provides a pseudomonas aeruginosa genetic engineering bacterium for high yield of phenazine-1-carboxylic acid, in particular to a pseudomonas aeruginosa (Pseudomonas chlororaphis) Qlu-1 genome phzO gene, degU gene, tctB gene and hppA gene which are knocked out to obtain pseudomonas aeruginosa genetic engineering bacterium QPCA delta DTH, wherein pseudomonas aeruginosa Qlu-1 is disclosed in Chinese invention patent CN 112126611B, the preservation unit name is China center for typical culture collection, the preservation address is China, university of Wuhan, the preservation date is 2020 month 08, and the preservation number is CCTCC NO: m2020108.

In a second aspect, the invention provides a construction method of a pseudomonas aeruginosa genetic engineering bacterium for high yield of phenazine-1-carboxylic acid, which takes pseudomonas aeruginosa (Pseudomonas chlororaphis) Qlu-1 with phenazine-1-carboxylic acid synthesis capability as an original strain, firstly knocks out a modified gene phzO to obtain a strain QPCA, so that phzO enzyme for catalyzing PCA to be converted into 2-hydroxy phenazine in the strain is inactivated, the strain specifically accumulates PCA, and then knocks out degU, tctB, hppA genes with negative regulation function on a strain QPCA genome to construct the pseudomonas aeruginosa genetic engineering bacterium QPCA delta DTH for high yield of phenazine-1-carboxylic acid.

Further, the knockdown method of phzO gene is as follows:

i. amplifying upstream and downstream homology arms of the phzO gene fragment; connecting upstream and downstream homologous arms by adopting a fusion PCR method and inserting the same into a pK18mobsacB plasmid to obtain a phzO gene recombinant plasmid;

ii. Introducing the phzO gene recombinant plasmid into escherichia coli S17-1 (lambda), and performing parent hybridization culture with pseudomonas aeruginosa Qlu-1 to introduce the phzO gene recombinant plasmid into pseudomonas aeruginosa Qlu-1;

and iii, screening positive clones to obtain the strain QPCA.

Further, the sequence of the phzO gene is shown as SEQ ID NO. 1;

the primers for amplifying the upstream homology arm of the phzO gene fragment comprise phzO-F1 and phzO-R1, and the sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3;

the primers for amplifying the downstream homology arm of the phzO gene fragment comprise phzO-F2 and phzO-R2, and the sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

the sequence of the upstream and downstream fusion fragment of the phzO gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID NO. 6.

Further, the knockout method of degU gene is as follows:

(1) Extracting a QPCA genome by using the kit;

(2) Searching the sequenced pseudomonas aeruginosa Qlu-1 genome sequence for the degU gene and the upstream and downstream sequences thereof, and fishing and connecting the upstream and downstream fragments degU-ud of the degU gene by PCR;

(3) Constructing degU knockout plasmid pK18-degU-ud by connecting degU-ud fragment with plasmid pK18 mobasacb through a seamless cloning technology;

(4) Introducing pK18-degU-ud into E.coli S17-1 (lambda) by heat shock transformation;

(5) Bacterial strain QPCA and escherichia coli S17-1 (lambda) are co-cultured and then coated on KB (A+K+) double-antibody plates, and single colony is selected;

(6) The QPCA knockout degU strain was co-screened by sucrose plate screening, photocopying screening and PCR screening.

Further, the sequence of the degU gene is shown as SEQ ID NO. 7;

the primers for amplifying the upstream homology arm of the degU gene fragment comprise degU-F1 and degU-R1, and the sequences are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9;

the primers for amplifying the downstream homology arm of the degU gene fragment comprise degU-F2 and degU-R2, and the sequences are respectively shown as SEQ ID NO.10 and SEQ ID NO. 11;

the sequence of the upstream and downstream fusion fragment of the degU gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID NO. 12.

Further, the knockout method of the tctB gene is the same as that of the degU gene, and the sequence of the tctB gene is shown in SEQ ID NO. 13;

the primer for amplifying the upstream homology arm of the tctB gene fragment comprises tctB-F1 and tctB-R1, and the sequences are respectively shown as SEQ ID NO.14 and SEQ ID NO. 15;

the primer for amplifying the downstream homology arm of the tctB gene fragment comprises tctB-F2 and tctB-R2, and the sequences are respectively shown as SEQ ID NO.16 and SEQ ID NO. 17;

the sequence of the fusion fragment on the upstream and downstream of the tctB gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID NO. 18.

Furthermore, the method for knocking out the hppA gene is the same as that for knocking out the degU gene, and the sequence of the hppA gene is shown as SEQ ID NO. 19;

the primers for amplifying the upstream homology arm of the hppA gene fragment comprise hppA-F1 and hppA-R1, and the sequences are respectively shown as SEQ ID NO.20 and SEQ ID NO. 21;

the primers for amplifying the downstream homology arm of the hppA gene fragment comprise hppA-F2 and hppA-R2, and the sequences are respectively shown as SEQ ID NO.22 and SEQ ID NO. 23;

the sequence of the fusion fragment of the hppA gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID NO. 24.

In a third aspect, the invention also provides an application of the pseudomonas aeruginosa genetically engineered bacterium for high yield of phenazine-1-carboxylic acid, in particular to a method for producing phenazine-1-carboxylic acid by fermenting the pseudomonas aeruginosa genetically engineered bacterium QPCA delta DTH.

The invention has the beneficial effects that:

according to the invention, phzO, degu, tctB and hppA genes of the pseudomonas aeruginosa Qlu-1 are knocked out to obtain a pseudomonas aeruginosa genetic engineering strain with high phenazine-1-carboxylic acid yield, the fermentation yield of the phenazine-1-carboxylic acid with high yield is improved from 270.4mg/L to 4787.2mg/L, and an important foundation is laid for the large-scale production of the phenazine-1-carboxylic acid.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is an electrophoretogram of the mutant plasmid pK18-degU-ud construction in example 2.

FIG. 2 is a diagram of a two-resistance panel screen for the parent hybridization of the degU gene in example 2.

FIG. 3 is a photograph of a double-exchanged positive monoclonal of the degU gene screened in example 2.

FIG. 4 is a PCR validation graph of QPCA knockout strain degU in example 2.

FIG. 5 is a graph showing the results of HPLC detection of Pseudomonas aeruginosa Qlu-1 and strain QPCA fermenters in the experimental example.

FIG. 6 is a graph showing the comparison of the production of phenazine-1 carboxylic acid by each strain in the test examples.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

The starting strain Pseudomonas aeruginosa (Pseudomonas chlororaphis) Qlu-1 used in the following examples was selected from the root of vegetable greenhouse pepper in the city of Weifang and was deposited in China center for type culture Collection, with a deposit address of China, university of Wuhan, and with a deposit number of CCTCC NO: m2020108.

The sequences used in the following examples are shown in Table 1.

Table 1 sequences used in the embodiments

EXAMPLE 1 obtaining Strain QPCA

The strain QPCA is obtained by knocking out the modified gene phzO of pseudomonas aeruginosa Qlu-1, and the knocking out method is as follows:

i. amplifying upstream and downstream homology arms of the phzO gene fragment; connecting upstream and downstream homologous arms by adopting a fusion PCR method and inserting the same into a pK18mobsacB plasmid to obtain a phzO gene recombinant plasmid;

ii. Introducing the phzO gene recombinant plasmid into escherichia coli S17-1 (lambda), and performing parent hybridization culture with pseudomonas aeruginosa Qlu-1 to introduce the phzO gene recombinant plasmid into pseudomonas aeruginosa Qlu-1;

and iii, screening positive clones to obtain the strain QPCA.

Example 2 obtaining of Pseudomonas aeruginosa genetically engineered bacterium QPCA DeltaD

The degU gene with negative regulation and control function is knocked out on the bacterial strain QPCA genome, and the Pseudomonas aeruginosa genetic engineering bacterium QPCA delta D is constructed, and the knocking out method is as follows:

(1) Inoculating the strain QPCA into LB (A+) culture medium, extracting and shake culturing at 180rpm at 30 ℃ for overnight, extracting genome of the QPCA by using genome extraction kit, and preserving at-20 ℃ for standby.

(2) Searching for the degU gene and the upstream and downstream sequences thereof in the sequenced Pseudomonas aeruginosa Qlu-1 genome sequence, and using the Qlu-1 strain genome as a template to amplify the upstream fragment degU-u and the line downstream fragment degU-d of the degU gene by using the degU-F1/degU-R1 and the degU-F2/degU-R2 as primers respectively; and amplifying the upstream and downstream fusion fragments degU-ud of the degU by taking degU-u and degU-d as templates and degU-F1/degU-R2 as templates.

(3) The fusion fragment degU-ud is connected with knockout plasmid pK18 mobasacb by enzyme digestion to construct recombinant plasmid pK18-degU-ud.

FIG. 1 shows construction of an electrophoretogram of mutant plasmid pK18-Degu-ud, wherein column 1 shows amplification of the upstream homology arm of degU, column 2 shows amplification of the downstream homology arm of degU, column 3 shows DNA Ladder DL5000, and columns 4 and 5 show fusion fragments of the upstream and downstream arms of degU gene.

(4) pK18-degU-ud was introduced into E.coli S17-1 (lambda) by heat shock transformation.

(5) As shown in FIG. 2, the strain QPCA was subjected to a double-parent hybridization culture with E.coli S17-1 (lambda), and single colonies were selected after being plated on KB (A+K+) double-antibody plates.

(6) As shown in fig. 3 and 4, QPCA knockout degU strain is jointly screened by sucrose plate screening, photocopying screening and PCR screening, wherein column 1 in the left diagram (external primer detection) of fig. 4 is a blank control, column 2 is DNA Ladder, column 3 is wild strain genome as template amplified fragment, and column 4 is degU knockout strain genome as template amplified fragment; in the right panel of FIG. 4 (internal primer test), column 1 is blank control, column 2 is Degu knockout strain genome as template amplified fragment, column 3 is DNA Ladder, and column 4 is wild strain genome as template amplified fragment.

After fermentation, the phenazine-1 carboxylic acid accumulated in the fermentation broth was increased by HPLC detection, and the strain was designated as QPAC. DELTA.D.

Example 3 obtaining of Pseudomonas aeruginosa genetically engineered bacterium QPCA DeltaDT

Based on the Pseudomonas aeruginosa genetically engineered bacterium QPCA delta D in example 2, the tctB gene with negative regulation function on the genome is knocked out, the knocked-out method of the tctB gene is the same as that of the degU gene, the related gene sequences are shown in table 1, and the strain obtained after knocked-out is named QPCA delta DT.

Example 4 obtaining of Pseudomonas aeruginosa genetically engineered bacterium QPCA DeltaDTH

Based on the Pseudomonas aeruginosa genetically engineered bacterium QPCA delta DT of example 3, the hppA gene with negative regulation function on the genome is knocked out, the method for knocking out the hppA gene is the same as the method for knocking out the degU gene, the related gene sequences are shown in table 1, and the strain obtained after knocking out is named QPCA delta DTH.

Verification example

Pseudomonas aeruginosa Qlu-1, bacterial strain QPCA, pseudomonas aeruginosa genetically engineered bacteria QPCA delta D, QPCA delta DT and QPCA delta DTH are respectively inoculated into KB culture medium for fermentation, extracted by ethyl acetate and then subjected to HPLC detection, and the results are shown in figures 5 and 6.

As can be seen from FIG. 5, only phenazine-1 carboxylic acid and no 2-hydroxy-phenazine were accumulated in the QPCA strain. As can be seen from FIG. 6, the 48h yield of Pseudomonas aeruginosa Qlu-1 is only about 270mg/L, the 48h yield of the strain QPCA is about 408mg/L, and the 48h yields of Pseudomonas aeruginosa genetically engineered bacteria QPCA delta D, QPCA delta DT and QPCA delta DTH respectively reach about 1028.7mg/L, 1892.5mg/L and 4787.2 mg/L.

Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.

SEQUENCE LISTING

<110> Qilu university of industry

<120> Pseudomonas aeruginosa genetically engineered bacterium for high yield of phenazine-1-carboxylic acid, construction method and application thereof

<130> 2022

<160> 24

<170> PatentIn version 3.5

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ttctctacgg ctggaggcat atcgcaagtt gcagaaatct acgaagaaca attcagcggt 180

gaacacgacg acattctgac ttacgtacgc cccgacggtt acctggcctc ttctgcctat 240

atgcccccta gaaacaaaga agacttggcg ttgcgacgcc gcgcaatcac gtacgtctcg 300

caaaaaacct ggggcaccca ctgccgtaac ctggacatga tcgccagctt caccgtcggc 360

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gtgatgagga tcaacaagca gaacgccgag ggcatctgga tcagcggcgt caaaggcgtg 600

ggcacggtag caccgcagtc caatgaaata tttgttggca gcttgttccc cgcagcgccc 660

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gagattgtct cccagcctca cgccagcgcc tatgaccacc cgctcatatc caaaggtgaa 780

gaagccgagg cgatggtggt attcgataac gtgttcattc cacgctggcg aatcatggcg 840

gcgaacgtgc cggaactggc caacgccggc ttcttcagcc tgtggacctc atacagccat 900

tggtacacgc tcgtgcgcct ggaaaccaag gctgacctgt atgccggact ggccaaggtg 960

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gagcagatca gcgccatgcg catccgcgaa gacggcttgc tcgccgagcg cagcgccgcg 1260

gcctcggcga cccgcatgcg cctgctgatg gccacgctgc tggcggcgct ctgcggcctg 1320

ttcggggcga ttgtcgcggt gctgttcctg tccaagggca tcgtcgcccg ggtgcagcag 1380

gtgcaaggca acgcccagcg cctggccctc ggccaaccgc tgcgcccgca ggccccggaa 1440

caggacgaga tcggccagct cggcacccgc ctggtggagg ccgggcaact gctggccgag 1500

cgcgagcggg ccctgcgcga taacgaagaa cgcctgcggc t 1541

<210> 13

<211> 507

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 13

atggccgggc gcagcagggt cgtgctgtcg cagttggcga ttggcctggg gctgatcgcc 60

atcagcctgg tgctggtcat cggcgccttg cgctttccgc ccgagatggg cttcgtcatc 120

ctcggtgccc atgtctatcc ctgcgccgtc ggtgcttttc taggggccgt gggcctgctg 180

ttgagctacc aggcctgcac cggtggtttg cgcgagctgg ccgcatccgg cgacgacagc 240

gacgacagcg acgacagcgc ccagcccggc ggcaggctcg gggcggcctg ggtcaccgcg 300

ggcctggtgg cgattgccgt actgatcaac ctgatcggtt tcgtcctggc cgccggcttg 360

ctgtttgcct gttcggcgcg gggtttcggc agccggcgtc cgttgcgcga cctggccatt 420

ggcatcgccc tgaccctgcc gatctactgg ctgttcagtg ccgggctggg cgtggccctg 480

ccacccctgg tcaatgcctg gatctga 507

<210> 14

<211> 30

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 14

acgaattcct ggcgcagttc gccaacagca 30

<210> 15

<211> 30

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 15

ccatctcaaa gcagcccgac cttgaccagc 30

<210> 16

<211> 36

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 16

ggctgctttg agatggtcaa tgcctggatc tgatcc 36

<210> 17

<211> 39

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 17

gcgctctaga ctccttctga tacagcaggc tgtacaagg 39

<210> 18

<211> 1467

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 18

ctggcgcagt tcgccaacag caccaagggc gacccgaatg cgctgctggt ggtgggggcg 60

atcatggtca ccgcggtgga gcagaacaag ccgcagatca gcctcaagga cgtgacgccg 120

attgcccggt tgttcaccga atacaacgtg ctcgccgtgc gtgaggagtc gccctacaag 180

accctcgagg agctgctcaa ggacttcaag gccaatccgg ccagcatcaa gtggggcggt 240

ggctccaagg gttccatcga ccatatcggc atcgccgagc tggcgagcaa gatggaggtg 300

ccggtgaacc aggtgaacta cgttgcctat gccggcggcg gcgaagtggt ggccgcgacc 360

ctgggcgggc acatcacggt gatcaccggc ggttatgccg aactggccaa gtacgtgcag 420

tccaagcagt tccgcctgct cgccatcggc gctccggagc gcgttcccgg catcgatgcg 480

ccgaccctca aggagaaggg ctacgacgtg atcatcggca actggcgcgg tgtctatggc 540

gcggccaacc tcacggccga gcaacgcaag caagtcaccg acgcggtcct ggccgccacc 600

aacagcaagg tctggcagga caatgtgaaa gccaacgcct ggtcgccgag catcctcacc 660

ggcgatgagt tcggcaaatt cgtcgacgag gaacaccagc gcctgcgggc gatgctggtc 720

aaggtcgggc tgctttgaga tggtcaatgc ctggatctga tccgggccga aggaggtcac 780

acggtggaca ttctcttgaa cctggcgaca ggtttctccg cggcgctggc gccgatcaat 840

ctgctctggg gatttatcgg ctgcctgctg ggcacggcga tcggcgtatt gccggggatc 900

ggcccggcgc tgacggtggc cctgctgttg ccgatcaccg ccaaggtcga tcccaccggc 960

gcgctgatca tgttcgccgg catctattac ggcgcgcagt tcggtggctc gaccacctcg 1020

atcctgctca ataccccggg cgagtcgtcg tccatggtca ccgccctgga aggcaatctc 1080

atggcccgta acggccgcgc cggaccggcc ctggccaccg cggcgatcgg ctcgtttttc 1140

gccggcacca tcgccacggt gctgctgacc ctgttcgccc ccatcgtcgc catgctggcg 1200

ctgaagttcg gccccgcgga gtatttcgcg atcctggtgc tgtccttcac cacggtgtcg 1260

gcggtgctcg gtgcgtccat gctgcgcggt ttcgcctcgc tggggattgg cctgggcatt 1320

ggcctgatcg gcctcgactc gacctcgggc attgcccgct acaccctggg cgttcccgag 1380

ctggtcgatg gcatcgaagt ggtgctggtg gcggtcggcc tgtttgccgt gggggaggcc 1440

ttgtacagcc tgctgtatca gaaggag 1467

<210> 19

<211> 789

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 19

atgcgtaaac aactgatgat ccctgccctg ttggccctga gcgtggcgct ggcggcatgc 60

tccaccccgc ctaacgccaa cctggaaaac gcccggacca actactccgc cctgcaggcc 120

aatccgcagg ccaccaaggt ggcggccctg gaaaccaagg acgccagcga atggctggac 180

aaggccgaca aggcctacct gaacaaggag gacgaaaagc aggtcgacca actggcctac 240

ctgaccaacc agcggattga agtggccaag cagaccatcg ccctgcgcac cgcggaaacc 300

cagctcaagg acgccggcga ccagcgcgcc aaggcgcttc tcgatgcccg cgacgcgcag 360

atcaagcaac tgcaatcgag cctcaacgcc aagcagaccg accgcggtac cctggtgacc 420

ttcggtgacg tgctgttcgc caccaacaag gccgacctga agtccagcgg cctggtcaac 480

atcaacaagc tggcgcagtt cctgcgcgac aaccctgacc gcaaagtgat tgtcgaaggc 540

tacaccgaca gcaccggcag cgcggcttac aaccagtcgc tgtccgagcg tcgcgccact 600

tcggtacagg tcgccctgat caagatgggt gtcgatccgg cacgcatcgt cgcccagggt 660

tatggcaagc aatacccggt tgccgacaac ggcagcgttt ccgggcgcgc catgaaccgt 720

cgtgtggaag tcaccatttc caacgacaac cagccggtcg cgccacgttc gaccgtcagc 780

gtcaactaa 789

<210> 20

<211> 35

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 20

tcgaattcaa gcctcgggtc acagccaata accat 35

<210> 21

<211> 41

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 21

gcataatggt ttcgtccttt tatcgattgc gaatacgtgg g 41

<210> 22

<211> 37

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 22

aaggacgaaa ccattatgcc gtcagcgtca actaagc 37

<210> 23

<211> 31

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 23

tatctagagc tgcggccttc gccgatccac a 31

<210> 24

<211> 1335

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas chlororaphis)

<400> 24

aagcctcggg tcacagccaa taaccatcac tggaacggcc gtttcatttt caacgccctc 60

agcatgtcgt cactcagcgt tacggcgtcg gaaccgggat ggattttgat gtttttcagg 120

agttatccaa tggagttgaa gaccatgaag atcagcactg ccaaatcctc gtccacccct 180

ctgcgcgggc tgaaactggc cgcgctggct ctcggtagca gcctggtcct ggccggttgc 240

gccggtaatc cgccaagcga gcaatacgcg gtgacgcaat ctgcggtcaa cagcgcagtc 300

agcgcaggcg gtaccgaatt tgccgccgtg gaaatgaaat cggctcagga caagctcaag 360

caagccgagc tcgccatgca cgacaagaac tacgacgaag cccggcgcct ggccgagcag 420

gccgaatggg acgcccgcgt cgccgagcgc aaggctcagg cagccaaggc cgaacaggca 480

ttgaaggact ctcagaaagg tgttcaggaa ctacgtcagg aaggcatgcg cagcgtgcaa 540

tgagccgcgg gcgccgattc ccacgtattc gcaatcgata aaaggacgaa accattatgc 600

cgtcagcgtc aactaagccc ggccctgcgc actcaaccgc gcagccaata aaaagccccg 660

ccttgatcgg cggggctttt ttatggttct tcacggaatt ccgggaaaca cagaaacaag 720

aacgccgatt tgattgatga tgttcgtgcg agcaaacaca tctaaaaaac cggcgttctt 780

atgcacgata ttaatacttt ccttcctttt tgggagggct tttctgtcgt cacgatcaag 840

cctgatggcg atgccctaca gatcgatctg acgccccaca ccacccgact cccttcctgt 900

ggtggatgcc aaaagccctg ttcaaccact cacgagtatt gcgagcgagt cgttcgtgat 960

ttgcccattc tcggccgtgc agtgcgcctc agcgttttgc tcagacgtgt tggctgtcgt 1020

gactgcggca aacgcatgga gactgtcagt tggctggatc gctatgcccg tatgacgcgc 1080

cgcttggccg atgcggtgat tcaagcctgc gagcgccttc ccacgttaca cgtggctcaa 1140

ctgtttgggt tgcattggga caccgttcgg ctgctagagc gtcgcgcctt gcaaacggca 1200

ttgagcgatc tgccgaaggc gcaaccacgg cgtctggtga tggacgagtt cgctctgttc 1260

aagggccatc gttacgccag tgtggtgctg gatgcggata cacgacgagt gctgtggatc 1320

ggcgaaggcc gcagc 1335

Claims (9)

1. A pseudomonas aeruginosa genetic engineering bacterium for high-yield phenazine-1-carboxylic acid is characterized by specifically knocking out pseudomonas aeruginosaPseudomonas chlororaphis) The phzO gene, the degU gene, the tctB gene and the hppA gene in the Qlu-1 genome are obtained to obtain pseudomonas aeruginosa genetic engineering bacterium QPCA delta DTH, wherein pseudomonas aeruginosa Qlu-1 is preserved in China center for type culture Collection, the preservation address is China, the university of Wuhan, the preservation date is 2020, 05, 08, and the preservation number is CCTCC NO: m2020108;

the sequence of the phzO gene is shown as SEQ ID NO. 1;

the sequence of the degU gene is shown in SEQ ID NO. 7;

the sequence of the tctB gene is shown as SEQ ID NO. 13;

the sequence of the hppA gene is shown in SEQ ID NO. 19.

2. A method for constructing a genetically engineered Pseudomonas aeruginosa bacterium capable of producing phenazine-1-carboxylic acid in high yield as claimed in claim 1, wherein the genetically engineered Pseudomonas aeruginosa bacterium is prepared from a Pseudomonas aeruginosa bacterium having a phenazine-1-carboxylic acid synthesizing abilityCytophytePseudomonas chlororaphis) Qlu-1 is an original strain, a modified gene phzO is knocked out to obtain a strain QPCA, and degU, tctB, hppA genes with negative regulation and control effects are knocked out on a strain QPCA genome sequentially to construct a Pseudomonas aeruginosa genetic engineering strain QPCA delta DTH for high-yield phenazine-1-carboxylic acid.

3. The construction method according to claim 2, wherein the phzO gene knockout method is as follows:

i. amplifying upstream and downstream homology arms of the phzO gene fragment; connecting upstream and downstream homologous arms by adopting a fusion PCR method and inserting the same into a pK18mobsacB plasmid to obtain a phzO gene recombinant plasmid;

ii. Introducing the phzO gene recombinant plasmid into escherichia coli S17-1 (lambda), and performing parent hybridization culture with pseudomonas aeruginosa Qlu-1 to introduce the phzO gene recombinant plasmid into pseudomonas aeruginosa Qlu-1;

and iii, screening positive clones to obtain the strain QPCA.

4. The construction method according to claim 3, wherein the phzO gene has a sequence shown in SEQ ID NO. 1;

the primers for amplifying the upstream homology arm of the phzO gene fragment comprise phzO-F1 and phzO-R1, and the sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3;

the primers for amplifying the downstream homology arm of the phzO gene fragment comprise phzO-F2 and phzO-R2, and the sequences are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

the sequence of the upstream and downstream fusion fragment of the phzO gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown in SEQ ID NO. 6.

5. The method of construction of claim 2, wherein the method of knockout of the degU gene is as follows:

(1) Extracting a QPCA genome by using the kit;

(2) Searching the sequenced pseudomonas aeruginosa Qlu-1 genome sequence for the degU gene and the upstream and downstream sequences thereof, and fishing and connecting the upstream and downstream fragments degU-ud of the degU gene by PCR;

(3) Constructing degU knockout plasmid pK18-degU-ud by connecting degU-ud fragment with plasmid pK18 mobasacb through a seamless cloning technology;

(4) Introducing pK18-degU-ud into E.coli S17-1 (lambda) by heat shock transformation;

(5) Bacterial strain QPCA and escherichia coli S17-1 (lambda) are co-cultured and then coated on KB (A+K+) double-antibody plates, and single colony is selected;

(6) The QPCA knockout degU strain was co-screened by sucrose plate screening, photocopying screening and PCR screening.

6. The construction method according to claim 5, wherein the degU gene has a sequence shown in SEQ ID NO. 7;

the primers for amplifying the upstream homology arm of the degU gene fragment comprise degU-F1 and degU-R1, and the sequences are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9;

the primers for amplifying the downstream homology arm of the degU gene fragment comprise degU-F2 and degU-R2, and the sequences are respectively shown as SEQ ID NO.10 and SEQ ID NO. 11;

the sequence of the upstream and downstream fusion fragment of the degU gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown in SEQ ID NO. 12.

7. The construction method according to claim 2, wherein the tctB gene knockout method is the same as the degU gene knockout method, and the sequence of the tctB gene is shown in SEQ ID No. 13;

the primer for amplifying the upstream homology arm of the tctB gene fragment comprises tctB-F1 and tctB-R1, and the sequences of the primers are respectively shown as SEQ ID NO.14 and SEQ ID NO. 15;

the primer for amplifying the downstream homology arm of the tctB gene fragment comprises tctB-F2 and tctB-R2, and the sequences of the primers are shown as SEQ ID NO.16 and SEQ ID NO.17 respectively;

the sequence of the fusion fragment of the tctB gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID NO. 18.

8. The construction method according to claim 2, wherein the method of knocking out the hppA gene is the same as the method of knocking out the degU gene, and the sequence of the hppA gene is shown in SEQ ID No. 19;

the primers for amplifying the upstream homology arm of the hppA gene fragment comprise hppA-F1 and hppA-R1, and the sequences are respectively shown as SEQ ID NO.20 and SEQ ID NO. 21;

the primers for amplifying the downstream homology arm of the hppA gene fragment comprise hppA-F2 and hppA-R2, and the sequences are respectively shown as SEQ ID NO.22 and SEQ ID NO. 23;

the sequence of the fusion fragment of the hppA gene obtained by connecting the upstream and downstream homology arms by adopting a fusion PCR method is shown as SEQ ID No. 24.

9. The use of a genetically engineered pseudomonas aeruginosa for high yield of phenazine-1-carboxylic acid according to claim 1, wherein the phenazine-1-carboxylic acid is produced by QPCA Δdth fermentation using the genetically engineered pseudomonas aeruginosa.

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