CN107043730A - Engineering strain, Preparation method and use for producing the formamide of azophenlyene 1 - Google Patents
Engineering strain, Preparation method and use for producing the formamide of azophenlyene 1 Download PDFInfo
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Abstract
The invention provides a kind of engineering strain of the formamide of azophenlyene 1 (PCN) and its production and use.The engineering strain can be prepared by the following method：Knock out Pseudomonas chlororaphis (Pseudomonas chlororaphis) HT66 CCTCC NO:PykA genes in M 2013467 and its derivative, are produced.The preparation method of the engineering strain comprises the following steps：Design of primers, construction of recombinant plasmid and mutant strain screening.Compared with prior art, the present invention has following beneficial effect：Using brand-new regulating strategy, change endocellular metabolism material flow direction, PCN yield is greatly improved；The engineering strain is safe and reliable, and stability is good, can remarkably promote PCN industrial or agricultural application.
Description
Technical field
The present invention relates to a kind of engineering strain, the preparation method and its usage of high yield azophenlyene -1- formamides (PCN),
Belong to gene engineering technology field.
Background technology
Up to the present, scientist has discovered that the compound for including phenazine structure more than 6000 kinds, big absolutely in them
Majority is all chemical synthesis, isolated out of organism to be less than 100 kinds.But the azophenlyene being separated to out of organism
Plant, the ability of animal pathogeny bacterium such as antibacterium, fungi with wide spectrum more than compound.For example, phenazine-1-carboxylic acid (PCA) energy
Enough take-alls for suppressing to cause wheat root to corrode, azophenlyene -1- formamides then have aobvious to pathogens such as tomato droops
The inhibitory action of work, and some phenazene derivatives then show the purposes in field of medicaments, have there is increasing scholar
Studying its mechanism of action.Bacterium being originated from natural compound phenazine more, it is many in them under suitable conditions during artificial culture
Can largely synthesize and secrete azophenlyene and its derivative, yield milligrams per liter arrive gram per liter level.These premium properties all show
The great potential of azophenlyene and the like large-scale promotion application is shown.
The synthesis of azophenlyene and controlling gene are more guarded in pseudomonad.Early in the 1970's, the same position of radioactivity is used
Plain labelling method has been found that the biosynthesis pathway of azophenlyene and branched acid (Shikimic acid) approach have close relationship
(Turner J M,Messenger A J.Occurrence,biochemistry and physiology of phenazine
pigment production.Adv Microb Physiol,1986,27：211-275).By contrasting different azophenlyene producing strains
Composition sequence find that the synthesis of azophenlyene at least needs the gene of coding PhzA, PhzD, PhzE, PhzF and PhzG albumen.It is most
Azophenlyene synthetic gene cluster is also comprising an Arabic heptanone saccharic acid -7- phosphoric acid (3-deoxy-D-arabino- of coding 3- deoxidations-a-
Heptulosonate-7-phosphate, DAHP) synzyme PhzC gene, it can be catalyzed point a first step for the sour approach of tree,
Make DAHP dephosphorylations and then be cyclized into phenyl ring, through dehydration, hydrogenation reaction formation shikimic acid, further be modified to form chorismic acid.
PhzC can promote metabolite to be converted to azophenlyene compound direction, its reaction being catalyzed and fragrant cluster amino acid synthesis pathway
It is similar.
Whole azophenlyene locus except comprising it is a set of by the azophenlyene synthetic gene of 7 genomic constitution in addition to, also in the presence of complicated generation
Thank to regulated and control network.In pseudomonad, the regulation and control of azophenlyene secondary metabolite can be divided into transcriptional control (Transcriptional
) and post-transcriptional control (Post-transcriptional control) two parts control.Such as group's sensing regulating system,
The two genes are named as phzI/phzR in fluorescence and Pseudomonas chlororaphis, and located immediately at the upper of azophenlyene synthetic gene
Trip.Chinese patent ZL200610023459.9 by changing the expression of the sub- gacA genes of global regulation in P. aeruginosa bacteria M 18,
The yield of phenazine-1-carboxylic acid greatly improved.The patent ZL201310566864.5 that this problem is declared, it is false single by knocking out green pin
Controlling gene psrA or the rpeA gene that azophenlyene is synthesized in born of the same parents bacterium HT66, also significantly increases the yield of azophenlyene -1- formamides.By
Complicated in the metabolic regulation network in microbial cell, different controlling gene interactions only rely on change controlling gene, it is difficult to
Further improve the yield of secondary metabolite.
Patent of the present invention is intended to the structural gene by knocking out metabolic pathway, changes the metabolic fluxes of related substances in cell
To, so that the direction accumulation that mesostate is synthesized towards azophenlyene, so as to promote the synthesis of azophenlyene class secondary metabolite.
The content of the invention
For defect of the prior art, it is an object of the invention to provide the engineering strain of high yield PCN a kind of and its
Preparation method and purposes.
The present invention is achieved by the following technical solutions：
In a first aspect, being to have knocked out green pin the invention provides a kind of engineering strain of high yield azophenlyene -1- formamides
Pseudomonad (Psedunomonaschlororaphis) HT66CCTCC NO:In M2013467 and its derivative genome
PykA genes, are produced.
Preferably, the base sequence of the pykA genes is as shown in SEQ ID NO.1.
Preferably, the amino acid sequence of the corresponding protein of the pykA genes is as shown in SEQ ID NO.2.
Second aspect, the invention provides a kind of system of the engineering strain of high yield azophenlyene -1- formamides as the aforementioned
Preparation Method, including insertion mutation method and traceless knockout method.
Preferably, the insertion mutation method knocks out pykA genes and comprised the following steps：
S1, amplification pykA genetic fragments, and insert in pEX18Tc plasmids；
In the middle of S2, the amplification resistant gene such as kanamycins or gentamicin, insertion pykA genes, pykA genes are made to occur to insert
Enter mutation；
S3, by the pykA genes and HT66 genomes after mutation pykA genes occur homologous recombination, sieved according to resistance
Select positive colony, you can.
Preferably, the traceless knockout pykA gene approaches comprise the following steps：
A1, the upstream and downstream homology arm for expanding pykA genetic fragments；
A2, fusion DNA vaccine method connection upstream and downstream homology arm are simultaneously inserted in pK18mobsacB plasmids；
Homologous recombination occurs for A3, the upstream and downstream homology arm fragment of the pykA genes made on recombinant plasmid and HT66 genomes,
Go out positive colony using sucrose pressure and resistance screening, you can.
The third aspect, present invention also offers a kind of culture medium for being used to cultivate foregoing engineering strain, it includes
Following component：Glycerine, peptone, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate.
Preferably, described culture medium includes the following component counted by weight：
Fourth aspect, exists invention further provides a kind of engineering strain of high yield azophenlyene -1- formamides as the aforementioned
Produce the purposes in azophenlyene -1- formamides.
Wherein, condition of the engineering strain when preparing azophenlyene -1- formamides is：Aerobic culture；Temperature：28~
32 degrees Celsius；pH：6~8；200~350rpm of rotating speed..
The present invention general principle be：PykA is the gene of encoding pyruvate acid kinase, the phase in the microorganism of different genera
Contrast is more conservative.Pyruvate kinase is a key enzyme in glycolytic pathway, can be with the transfer of catalytic phosphatase group by phosphoric acid
Enol pyruvic acid (PEP) changes into pyruvic acid and circulated hence into TCA or other approach.Because PEP and pyruvic acid are in carefully
Intracellular sugar, the key position of amino acid and lipid metaboli, and PEP be one of initial substrate in shikimic acid route of synthesis (erythrose-
4- phosphoric acid (E4P) is another substrate), the end-product chorismic acid of shikimic acid pathway is the straight of azophenlyene -1- formamides (PCN) synthesis
Substrate is connect, therefore the reaction that PEP changes into pyruvic acid can be weakened by knocking out pykA genes, more carbon sources is flowed to shikimic acid way
Footpath, so as to improve PCN yield.
Therefore, compared with prior art, the present invention has following beneficial effect：
Using brand-new regulating strategy, change endocellular metabolism material flow direction, PCN yield is greatly improved；The base
Because engineered strain is safe and reliable, stability is good, can remarkably promote PCN industrial or agricultural application.
Brief description of the drawings
By reading the detailed description made with reference to the following drawings to non-limiting example, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is that (L1 is blank control, L2 for the electrophoretograms of pykA gene amplification products in different strains:With wild strain HT66
Genome be template, L3:Using mutant strain HT66 Δs pykA genome as template)；
Fig. 2 is wild strain HT66 and mutant strain HT66 Δs pykA growth curve comparison diagram；
Fig. 3 is that (L1 is blank control, L2 for the electrophoretograms of pykA gene amplification products in different strains:With bacterial strain P3 base
Because group is template, L3:Using mutant strain P3 Δs pykA genome as template)
Fig. 4 is bacterial strain P3 and mutant strain P3 Δs pykA growth curve comparison diagram；
Fig. 5 is wild strain HT66 and mutant strain HT66 Δs pykA PCN fermentation diagrams；
Fig. 6 is bacterial strain P3 and mutant strain P3 Δs pykA PCN fermentation diagrams.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that to the ordinary skill of this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Pseudomonas chlororaphis (Psedunomonas chlororaphis) HT66 of the present invention, the bacterial strain is in Chinese allusion quotation
Type culture collection (abbreviation CCTCC) preservation, depositary institution address is：Chinese Wuhan Wuhan Universitys, postcode is：
430072, preservation date is：On October 12nd, 2013, deposit number is：CCTCC NO∶M 2013467.Bacterial strain P3 passes through for HT66
Cross the PCN superior strains obtained after multiple mutation【Zhang Pingyuan, Peng Huasong, Shen Xuemei, wait the green pin of high yield azophenlyene -1- formamides
The mutagenesis of pseudomonad and genetic engineering breeding Shanghai communications university's journals:Agricultural sciences version, 2015,33 (2):90-94.】.
Embodiment 1
The present embodiment is related to one kind using HT66 genomes as template, and HT66 Δ pykA gene works are prepared using insertion mutation method
The method of journey bacterial strain, comprises the following steps：
1. design of primers
Using pykA gene orders as reference, design primer using primer 5 and synthesize, primer sequence is as follows, underscore table
Show restriction enzyme Hind III and Sac I restriction enzyme site.
pykA1:CCAAGCTTATGTCCGTCCGTCGTACCAA(SEQ ID NO.3)
pykA2:ACGAGCTCTCAGACCATTGGGTCGCCA(SEQ ID NO.4)
Using plasmid pBbB5k as template, design blocks the primer of that resistant gene (Kan) and synthesis, and primer sequence is as follows, under
Line represents restriction enzyme Xho I restriction enzyme site.
pBb-Kan-F:CCCTCGAGGGAATTGCCAGCTGGGGCGC SEQ ID NO.5
pBb-Kan-R:CCCTCGAGTCAGAAGAACTCGTCAAGAAG SEQ ID NO.6
2. insertion mutation construction of recombinant plasmid
Using HT66 genomes and pBbB5k plasmids as template, PCR points are passed through using pykA1/2 and pBb-Kan-F/R primers
Not Kuo Zeng pykA genes (1452bp) and Kan genes (1000bp), amplified production after purification, Kan gene PCR products are first placed in-
20 ° of refrigerators are preserved.PykA genes and pEX18Tc plasmids are subjected to double digestion, digestion system respectively using Hind III and Sac I
Gel extraction, 16 DEG C of connections and Transformed E .coli DH5a competent cells, sun is obtained by the screening of Tc (20ppm) resistant panel
Property clone.PEX-pykA plasmids are extracted, Xho I single endonuclease digestions are carried out respectively together with Kan genes, 16 DEG C connected after rubber tapping is reclaimed
Night simultaneously converts DH5a, obtains positive colony using the resistant panel screening recon containing Tc (20ppm), Kan (50ppm), extracts
The plasmid pEX-pykA-Kan of acquisition, is transferred to E.coli SM10 competent cells.
3. insertion mutation bacterial strain screening
The SM10 and bacterial strain HT66 that are transferred to pEX-pykA-Kan plasmids are subjected to biparent cross, pass through Tc (150ppm), Kan
(50ppm), Sp (100ppm) LB resistant panels carry out single-swap screening, containing 15% sucrose and Kan (50ppm), Sp
The LB flat boards of (100ppm) carry out double crossing over screening, and external primers (pykA1/2) PCR checkings carry genome amplification and send sequencing, most
Insertion mutation strain HT66 Δs pykA is obtained afterwards.
Enter performing PCR checking using pykA1 and pykA2 primers, insertion mutation bacterial strain is amplifiable to be gone out to be about 2500bp fragment
(pykA and kan gene sizes sum), and the wild strain not knocked out is then amplifiable goes out to be about 1500bp fragment (pykA sizes
For 1452bp), as a result as shown in Figure 1.In Fig. 1：M is Marker, and L1 is blank control, L2:Using bacterial strain HT66 genome as
Template, L3:Genome using bacterial strain HT66 Δs pykA is template.
By Liquid Culture, bacterial strain HT66 and HT66 Δ pykA growth curves are determined as shown in Figure 2.As a result it is shown in HT66
Middle knockout pykA genes do not interfere with its growth, show that the stability of the genetic engineering bacterium is good, are conducive to entering the bacterium traveling one
Step transformation.
Embodiment 2
The present embodiment is related to a kind of preparation of high yield PCN engineering strain, and starting strain P3 is Pseudomonas chlororaphis
The bacterial strain for the high yield PCN that HT66 is obtained after multiple mutation.Specifically include following steps：
1st, design of primers：
Using pykA gene upstream and downstream sequences as reference, knock out primer using the designs of primer 5 and synthesize, primer sequence is such as
Under (underscore represents XbaI and HindIII restriction enzyme sites)：
pykAF1:CCGGGGATCCTCTAGAAAGATCGTTACAACGCGGTCG(SEQ ID NO.7)
pykAR1:TGGTACGACGGACGGACATG(SEQ ID NO.8)
pykAF2:TGTCCGTCCGTCGTACCAACCCAATGGTCTGAGCCACC(SEQ ID NO.9)
pykAR2:GGCCAGTGCCAAGCTTCGAGTTCGGTTCCAGCCTG(SEQ ID NO.10)
2nd, construction of recombinant plasmid：
Using P3 genomes as template, above and below pykA is expanded by PCR using pykAF1, pykAR1 and pykAF2, pykAR2
Homology arm fragment is swum, PCR primer after purification, is connected the upstream and downstream homology arm using In-Fusion HD Cloning Kit methods
It is connected on the pk18mobsacB plasmids for carried out digestion and Transformed E .coli DH5a, is screened by blue hickie, hickie bacterium colony
Sequencing is sent after entering performing PCR checking using primer pykAF1 and pykAR2.
3rd, mutant strain is screened：
Correct plasmid pK18-pykA will be sequenced to convert to E.coli S17, is combined transfer with bacterial strain P3, passes through
Kan and Amp resistant panels single-swap is screened, 15% (w/v) sucrose plate double crossing over screening, and external primers PCR checkings carry genome
Sequencing is sent in amplification, is finally obtained bacterial strain P3 pykA knockout mutant strains, is named as P3 Δ pykA, is drawn using pykAF1 and pykAR2
Thing PCR is expanded, and knock-out bacterial strain is amplifiable to be gone out to be about 1100bp pykA upstream and downstream homology arm fragments, and the wild strain not knocked out
It is then amplifiable to go out to be about 2500bp fragment (pykA sizes are 1452bp), show pykA gene successful knockouts, as a result such as Fig. 3 institutes
Show.In Fig. 3, L1 is blank control, L2:Using bacterial strain P3 genome as template, L3:Using mutant strain P3 Δs pykA genome as
Template.
By carrying out Liquid Culture in KB culture mediums, while bacterial strain P3 and P3 Δ pykA growth curves are determined, such as Fig. 4 institutes
Show.As a result show, pykA genes are knocked out in bacterial strain P3 and do not interfere with its growth, show this engineering strain stability very
It is good, be conducive to its industrial applications.
Embodiment 3
The present embodiment is related to a kind of engineering strain synthesis PCN prepared by utilization embodiment 1 method, including following step
Suddenly：
Bacterial strain HT66 Δs pykA and HT66wt (wild strain HT66 is used as control) are lived on KB solid plates respectively respectively
Change twice, be then connected to 5mLKB fluid nutrient medium cultures, culture to logarithmic phase is forwarded to 2% ratio and trained equipped with 60mLKB
The triangular flask for supporting base is fermented.Fermentation condition is 28 DEG C, 180rpm.Timing sampling, bacterial strain PCN yield is determined with HPLC.
HPLC testing conditions：
Mobile phase is acetonitrile -25mM ammonium acetates, and chromatographic column is (5 μm of WondaSilC18-WRreversephasecolumn；
4.6 × 250mm, Shimadzu, Japan), Detection wavelength 254nm, testing conditions：0-2min, 8% acetonitrile -92%25mM acetic acid
Ammonium, 2-20min, acetonitrile concentration rises to 60% from 8%, acetic acid ammonium concentration from 92% drop to 40%, 20-21min, 8% acetonitrile-
92%25mM ammonium acetates.
Bacterial strain HT66 and bacterial strain HT66 Δs pykA PCN yield is determined respectively, as a result as shown in Figure 5.From figure, knock out
PykA genes can significantly improve PCN yield, and control strain HT66 PCN yield is 552mg/L, and mutant strain its PCN in 48h
Yield is 1038mg/L, compared to improving 88%.As can be seen here, pykA genes being knocked out in Pseudomonas chlororaphis HT66 can have
Effect changes the flow direction of material, is conducive to accumulating PCN.
Embodiment 4
The present embodiment is related to a kind of engineering strain production PCN prepared by utilization embodiment 2 method, including following step
Suddenly：
Bacterial strain P3 Δ pykA and P3 (control) are activated on KB solid plates respectively first, 5mL KB are then connected to
Fluid nutrient medium culture, after it grows into logarithmic phase being seeded to the triangular flask equipped with 60mLKB culture mediums respectively is fermented.
Initial inoculum OD600 is 0.02.Fermentation condition is 28 DEG C, 180rpm.Timing sampling, determines bacterial strain P3 Δs pykA and P3 respectively
PCN yield in zymotic fluid, as a result as shown in Figure 6.
From figure, PCN yield can be significantly improved by knocking out pykA genes, and in 36h, its PCN yield is 3594mg/L,
Compared with strain P3 PCN yield (1923mg/L) is compareed under similarity condition, 71.3% is improved.The result shows, false in green pin
PykA genes are knocked out in monad P3 can effectively reallocate the flow directions of cell carbon and nitrogen sources, it is flowed to shikimic acid pathway, enter
And flow to PCN synthesis.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (10)
1. a kind of engineering strain of high yield azophenlyene -1- formamides, it is characterised in that knocked out Pseudomonas chlororaphis
(Pseudomonas chlororaphis)HT66 CCTCC NO:PykA genes in M2013467 and its derivative genome,
Produce.
2. the engineering strain of high yield azophenlyene -1- formamides as claimed in claim 1, it is characterised in that the pykA bases
The base sequence of cause is as shown in SEQ ID NO.1.
3. the engineering strain of high yield azophenlyene -1- formamides as claimed in claim 1, it is characterised in that the pykA bases
The amino acid sequence of the corresponding protein of cause is as shown in SEQ ID NO.2.
4. a kind of preparation method of the engineering strain of high yield azophenlyene -1- formamides as described in claim 1,2 or 3, its
It is characterised by, the method for knocking out the pykA genes is insertion mutation method or traceless knockout method.
5. the preparation method of the engineering strain of high yield azophenlyene -1- formamides as claimed in claim 4, it is characterised in that
The insertion mutation method knocks out pykA genes and comprised the following steps：
S1, amplification pykA genetic fragments, and insert in pEX18Tc plasmids；
In the middle of S2, the amplification resistant gene such as kanamycins or gentamicin, insertion pykA genes, pykA genes are made to occur insertion prominent
Become；
S3, by the pykA genes and HT66 genomes after mutation pykA genes occur homologous recombination, gone out according to resistance screening
Positive colony, you can.
6. the preparation method of the engineering strain of high yield azophenlyene -1- formamides as claimed in claim 4, it is characterised in that
The traceless knockout pykA genes comprise the following steps：
A1, the upstream and downstream homology arm for expanding pykA genes；
A2, fusion DNA vaccine method connection upstream and downstream homology arm are simultaneously inserted in pK18mobsacB plasmids；
Homologous recombination occurs for A3, the upstream and downstream homology arm fragment of the pykA genes made on recombinant plasmid and HT66 genomes, utilizes
Sucrose pressure and resistance screening go out positive colony, you can.
7. a kind of culture medium for being used to cultivate the engineering strain described in claim 1, it is characterised in that including such as the following group
Point：Glycerine, peptone, yeast extract, magnesium sulfate, dipotassium hydrogen phosphate.
8. culture medium as claimed in claim 7, it is characterised in that including the following component counted by weight：
9. a kind of engineering strain of high yield azophenlyene -1- formamides as described in any one in claims 1 to 3 is in production
Purposes in azophenlyene -1- formamides.
10. purposes as claimed in claim 9, wherein, condition of the engineering strain when preparing azophenlyene -1- formamides
For：Aerobic culture；Temperature：28~32 degrees Celsius；pH：6~8；200~350rpm of rotating speed.
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| PCT/CN2017/098480 WO2018036479A1 (en) | 2016-08-22 | 2017-08-22 | Genetically engineered strain of high yield phenazine-1-carboxamide, construction method for same, and uses thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036479A1 (en) * | 2016-08-22 | 2018-03-01 | 上海交通大学 | Genetically engineered strain of high yield phenazine-1-carboxamide, construction method for same, and uses thereof |
| CN109142578A (en) * | 2018-09-12 | 2019-01-04 | 中国人民解放军军事科学院军事医学研究院 | The method for detecting two kinds of gene pairs target metabolite regulation relationships in biological cell |
| CN112111440A (en) * | 2020-09-25 | 2020-12-22 | 齐鲁工业大学 | Genetically engineered bacterium for producing phenazine-1-formamide and preparation method and application thereof |
| CN114057657A (en) * | 2021-12-14 | 2022-02-18 | 上海农乐生物制品股份有限公司 | Purification method of phenazine-1-formamide |
| CN115181754A (en) * | 2022-06-22 | 2022-10-14 | 齐鲁工业大学 | Pseudomonas chlororaphis genetic engineering bacterium for high yield of phenazine-1-carboxylic acid and construction method and application thereof |
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| WO2018036479A1 (en) * | 2016-08-22 | 2018-03-01 | 上海交通大学 | Genetically engineered strain of high yield phenazine-1-carboxamide, construction method for same, and uses thereof |
| CN109142578A (en) * | 2018-09-12 | 2019-01-04 | 中国人民解放军军事科学院军事医学研究院 | The method for detecting two kinds of gene pairs target metabolite regulation relationships in biological cell |
| CN109142578B (en) * | 2018-09-12 | 2021-08-10 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting regulation and control relation of two genes to target metabolite in biological cell |
| CN112111440A (en) * | 2020-09-25 | 2020-12-22 | 齐鲁工业大学 | Genetically engineered bacterium for producing phenazine-1-formamide and preparation method and application thereof |
| CN112111440B (en) * | 2020-09-25 | 2022-03-04 | 齐鲁工业大学 | Genetically engineered bacterium for producing phenazine-1-formamide and preparation method and application thereof |
| CN114057657A (en) * | 2021-12-14 | 2022-02-18 | 上海农乐生物制品股份有限公司 | Purification method of phenazine-1-formamide |
| CN114057657B (en) * | 2021-12-14 | 2024-03-22 | 无锡秋可生物科技有限公司 | Purification method of phenazine-1-carboxamide |
| CN115181754A (en) * | 2022-06-22 | 2022-10-14 | 齐鲁工业大学 | Pseudomonas chlororaphis genetic engineering bacterium for high yield of phenazine-1-carboxylic acid and construction method and application thereof |
| CN115181754B (en) * | 2022-06-22 | 2023-05-19 | 齐鲁工业大学 | Pseudomonas aeruginosa genetically engineered bacterium for high-yield phenazine-1-carboxylic acid and construction method and application thereof |
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