Team:Bielefeld-Germany/Project/Phenazine

From 2013.igem.org

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[[File:IGEM Bielefeld 2013Phenazine-3D-balls-B.png|300px|thumb|left| '''Figure 1:''' Molecular structure of phenazine.]]  
[[File:IGEM Bielefeld 2013Phenazine-3D-balls-B.png|300px|thumb|left| '''Figure 1:''' Molecular structure of phenazine.]]  
<p align="justify">
<p align="justify">
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Phenazines are water-soluble secondary metabolites secreted by some bacterial species. They are mostly known for their pathogenic properties, attributed to their ability to undergo redox transformations. Bacteria are also able to use this substance as a mediator for electron shuttling, which leads to an efficiency gain if a phenazine-producing strain is present in a MFC.
+
Phenazines are water-soluble secondary metabolites secreted by some bacterial species. Almost all their properties can be attributed to their ability to undergo redox transformations. Some soil-born bacteria commonly produce phenazine-1-carboxylic acid ([http://www.ebi.ac.uk/chebi/chebiOntology.do?chebiId=CHEBI:62412 PCA])) against fungal root decease. (Niknejad Kazempour, [http://www.agriculturaits.czu.cz/pdf_files/vol_42_4_pdf/kazempour.pdf 2009]) Bacteria are also able to use this substance as a mediator for electron shuttling, which leads to an efficiency gain if a phenazine-producing strain is present in a MFC.
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<br>  
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<br>
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Initially phenazine overproduction seemed to be a promising approach to achieve higher electron transfer, but considering biosafety aspects and an extremely complicated cloning procedure we abandoned this sub-project on early stages and searched for alternatives.  
+
Problems arise with the different phenazine producing host strains. Most of them are classified as pathogens and therefore above the risk group, which is suitable for our lab.
-
We concentrated us on an overproduction of environmentally non-invading riboflavin and the overexpression of the glycerol dehydrohenase gldA. This has proved to be an excellent decision (See sections [https://2013.igem.org/Team:Bielefeld-Germany/Project/Riboflavine riboflavin] and [https://2013.igem.org/Team:Bielefeld-Germany/Project/GldA GldA] on our Wiki for more information).  
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Moreover the team’s own risk assessment settles that no strain above the risk group one should be used. Hence, all experiments carried out in this section were soluble done with
 +
''Pseudomonas fluorescence'' which is classified as risk group 1.
 +
<br>
 +
Initially PCA overproduction seemed to be a promising approach to achieve higher electron transfers. Because of the extremely complicated cloning procedure as well as the good advice of the consulted MFC expert [https://2013.igem.org/Team:Bielefeld-Germany/Human_Practice/Experts Dr. Falk Harnisch], we abandoned this sub-project on early stages and searched for alternatives. We concentrated on an overexpression of riboflavin, which serves as an electrone shuttle itself and of the glycerol dehydrohenase gldA., which increases NADH concentration. (See sections [https://2013.igem.org/Team:Bielefeld-Germany/Project/Riboflavine riboflavin] and [https://2013.igem.org/Team:Bielefeld-Germany/Project/GldA gldA] on our Wiki for more information).  
Still, quite a lot of work was invested into investigation of phenazine functions and synthesis in bacteria, of so we present a short theoretical overview of this topic.
Still, quite a lot of work was invested into investigation of phenazine functions and synthesis in bacteria, of so we present a short theoretical overview of this topic.
-
The only entry in the Labjournal on phenazine is found [https://2013.igem.org/Team:Bielefeld-Germany/Labjournal/July here] in Week 13.
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The only entry in the labjournal on phenazine is found [https://2013.igem.org/Team:Bielefeld-Germany/Labjournal/July here] in Week 13.
</p>
</p>
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Phenazines comprise a large group of heterocyclic nitrogen-containing substances, having a tricyclic ring in its core. They are substituted at different points around their rings, which alters water solubility and other properties. After discovery of naturally occurring phenazines, more than 6000 were chemically synthesized. (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/16719720 2006]). [http://en.wikipedia.org/wiki/Neutral_red Neutral red], a well-known pH-indicator, also belongs to this class.
Phenazines comprise a large group of heterocyclic nitrogen-containing substances, having a tricyclic ring in its core. They are substituted at different points around their rings, which alters water solubility and other properties. After discovery of naturally occurring phenazines, more than 6000 were chemically synthesized. (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/16719720 2006]). [http://en.wikipedia.org/wiki/Neutral_red Neutral red], a well-known pH-indicator, also belongs to this class.
<br><br>
<br><br>
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Phenazine derivatives were one of the first secondary bacterial metabolites that were consequently studied since their discovery in 1859. The first substance of this family, a blue pigment of ''Pseudomonas aeroguinosa'', was called [http://en.wikipedia.org/wiki/Pyocyanine «pyocyanine»]. It had antimicrobial properties that raised further interest in these substances.
+
Phenazine derivatives were one of the first secondary bacterial metabolites that were consequently studied since their discovery in 1859. The first substance of this family, a blue pigment of ''Pseudomonas aeroguinosa'', was called [http://en.wikipedia.org/wiki/Pyocyanine «pyocyanine»] (short PYO). It had antimicrobial properties that raised further interest in these substances.
-
Phenazines are produced by many bacterial species, but they are mostly studied in representatives of fluorescent pseudomonads. A single bacterial strain usually produces two or more species-specific phenazines, except ''Pseudomonas fluorescens'', that is known to produce only PCA ([http://www.ebi.ac.uk/chebi/chebiOntology.do?chebiId=CHEBI:62412 phenazine-1-carboxylic acid]). (Mavrodi et al., [http://www.ncbi.nlm.nih.gov/pubmed/11591691 2001])  
+
Phenazines are produced by many bacterial species, but they are mostly studied in representatives of fluorescent pseudomonads. A single bacterial strain usually produces two or more species-specific phenazines, except ''Pseudomonas fluorescens'' 2-79, that is known to produce only PCA, a broad-spectrum antibiotic, which is a casual agent active against a variety of root pathogens (Mavrodi et al., [http://www.ncbi.nlm.nih.gov/pubmed/11591691 2001]).
<br><br>
<br><br>
-
Biologically phenazines are known for their broad-spectrum of antibiotical, antiparazitar and even anti-malaria properties (Handelsmann et al., [http://www.plantcell.org/content/8/10/1855.long 1996]). They are highly associated with pathogenicity of bacterial species.  A broad-range, non-specific mechanism of phenazine action was firstly thought to be aimed against an essential metabolic pathway (Leo C. Vining,  [http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.44.100190.002143 1990]), but potentiometric measurements on PYO discovered a redox system, that was formed between its reduced and oxidised derivatives.
+
Biologically phenazines are known for their broad-spectrum of antibiotical, antiparasaticand even anti-malaria properties (Handelsmann et al., [http://www.plantcell.org/content/8/10/1855.long 1996]).
-
Consequently it was shown, that almost all properties of phenazines could be attributed to their ability to undergo redox transformations (Muller, [http://www.ncbi.nlm.nih.gov/pubmed/8541351 1995]). This discovery brought up the idea, that these molecules can be used as an electron shuttle in a MFC. Indeed, it was shown that external addition of phenazines, both PCA and PCN ([http://www.ebi.ac.uk/chebi/searchId.do?chebiId=62240 phenazine-1-carboxamide]) alike, causes a significant raise of current flow (The Hai Pham, [http://www.ncbi.nlm.nih.gov/pubmed/18688612 2008]). Surprising, a mixed culture of phenazine-secreting and phenazine non-secreting strains showed similar results, proving that phenazine can be used for redox cycling not only by its producer (Hernandez et al. [http://www.ncbi.nlm.nih.gov/pubmed/14766572 2004]).
+
Consequently it was shown, that almost all properties of phenazines could be attributed to their ability to undergo redox transformations (Muller, [http://www.ncbi.nlm.nih.gov/pubmed/8541351 1995]). This discovery brought up the idea, that these molecules can be used as an electron shuttle in a MFC. Indeed, it was shown that external addition of phenazines, both PCA and PCN ([http://www.ebi.ac.uk/chebi/searchId.do?chebiId=62240 phenazine-1-carboxamide]) alike, causes a significant raise of current flow (The Hai Pham, [http://www.ncbi.nlm.nih.gov/pubmed/18688612 2008]). Surprisingly, a mixed culture of phenazine-secreting and phenazine non-secreting strains showed similar results, proving that phenazine can be used for redox cycling not only by its producer (Hernandez et al. [http://www.ncbi.nlm.nih.gov/pubmed/14766572 2004]).
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A group of soilborne, root-desease controlling microorganisms, such as ''Pseudomonas fluorescens'' and ''Pseudomonas chlororaphis'', are known to produce the phenazine compounds PCA and PCN. They belong to safety class S1, so we saw our chance to try a heterologous expression, taking the coding sequences from one of the known phenazine producers. There were already reports of successful phenazine expression in ''E.Coli'' (MacDonald et al. [http://www.ncbi.nlm.nih.gov/pubmed/11562236 2001])
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<br><br>
 +
Many strains of Pseudomonas fluorescens, a common soil inhabitant, are known to produce the phenazine compound PCA. The phenazine synthesis pathway was studied in detail. The seven-gene cluster [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 ''phzABCDEFG''], 8505 bp long, was firstly identified in ''Pseudomonas fluorescens'' 2-79.
<br><br>
<br><br>
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The phenazine synthesis is a branch-off of a [http://en.wikipedia.org/wiki/Shikimic_acid| shikimic acid] pathway that delivers aromatic acids. It was discovered and researched in detail for ''Pseudomonas aeroguinosa'' and ''Pseudomonas fluorescens'' 2-79. The seven-gene cluster [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 ''phzABCDEFG''] was identified, that was to almost 95% identical in both organisms.</p>  
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<p align="justify">Research on phenazine diversity and genetic evolution, (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/20008172 2010]) stated, that many species of fluorescent pseudomonas have the corresponding sequences and are able to produce phenazines, so we expected that similar genes are found in the ''Pseudomonas fluorescens'' 50090 that we obtained from [http://www.dsmz.de DSMZ].</p>  
[[File:IGEM Bielefeld 2013 phz genes table.png|thumb|left|400px|'''Figure 2''': Comparison of sequences between enzymes in phenazine cluster and other proteins with known function, according to research of [http://www.ncbi.nlm.nih.gov/pubmed/11562236 MacDonald et al. 2001.]]]
[[File:IGEM Bielefeld 2013 phz genes table.png|thumb|left|400px|'''Figure 2''': Comparison of sequences between enzymes in phenazine cluster and other proteins with known function, according to research of [http://www.ncbi.nlm.nih.gov/pubmed/11562236 MacDonald et al. 2001.]]]
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<p align="justify">Also research on phenazine diversity and genetic evolution, (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/20008172 2010]) brought us to the thought, that many species of fluorescent pseudomonas have the corresponding sequences and are able to produce phenazines. We have already thought over a cloning strategy, but the fact, that phenazines are highly associated with pathogenicity of bacterial species, was a serious concern about biological safety of constructing a strain, overexpressing phenazines. We were uncertain, if this is an appropriate option and what consequences can we await.
 
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==Genetic Approach==
==Genetic Approach==
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[[File:IGEM Bielefeld 2013 Phenazines genes.png|thumb|center|500px|'''Figure 3:''' Phenazine synthesis cluster, comprising an autoinducer synthase (phzI) gene, positive regulator protein (phzR) and coding [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 ''phzABCDEFG''] genes.]]
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[[File:IGEM Bielefeld 2013 Phenazines genes.png|thumb|center|500px|'''Figure 3:''' Phenazine synthesis cluster, comprising an autoinducer synthase (''phzI'') gene, positive regulator protein (phzR) and coding [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 ''phzABCDEFG''] genes.]]
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<br>
<p align="justify">
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A close look on the cluster revealed seven forbidden restriction sites, making a cloning in accordance with the iGEM-rules an ultimate challenge for a short-termed project.
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The coding cluster revealed seven forbidden restriction sites, making a cloning in accordance with the iGEM-rules an ultimate challengeUnfortunately, this step did never success. We  
-
At first, we still pursued the idea and tried to multiply the phenazine-producing cluster, a 8505 bp long fragment by PCR. Unfortunately, this step did never success.
+
varied different parameters and tried different enzymes, but we always saw a false priming and multiple amplificates. So we decided to look for applicable alternatives.  
-
We varied different parameters and tried different enzymes, but we always saw a false priming and multiple amplificates.
+
Riboflavin, a natural mediator seemed an alternative of choice and our decision was rewarded by successful results.
-
Considering all these arguments, we realized that this sub-project will be impossible to pursue in the time we had, and even if we succeed, a strain, producing a natural antibiotic acting as a mediator was very questionable from the environmental point of view. So we decided to look for applicable alternatives.  
+
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Riboflavin, a natural mediator that is not associated with any pathogenicity seemed an alternative of choice and our decision was rewarded by successful results.  
+
</p>
</p>
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==Results==
==Results==
<p align="justify">
<p align="justify">
-
There are no practical results in this section. The project was left on the side after unsuccessful attempts to amplify the phenazine-coding fragment from ''Pseudomonas fluorescens'' sp. The only entry in the Labjournal on phenazine is found [https://2013.igem.org/Team:Bielefeld-Germany/Labjournal/July here] in Week 13.
+
There are no practical results in this section. The project was left on the side after unsuccessful attempts to amplify the phenazine-coding fragment from ''Pseudomonas fluorescens'' sp. The only entry in the labjournal on phenazine is found [https://2013.igem.org/Team:Bielefeld-Germany/Labjournal/July here] in Week 13.
</p>
</p>
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* Mavrodi D.V. (2010) Diversity and evolution of the phenazine biosynthesis pathway. [http://www.ncbi.nlm.nih.gov/pubmed/20008172 ''Appl Environ Microbiol.:866-79''.]  
* Mavrodi D.V. (2010) Diversity and evolution of the phenazine biosynthesis pathway. [http://www.ncbi.nlm.nih.gov/pubmed/20008172 ''Appl Environ Microbiol.:866-79''.]  
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* Niknejad Kazempour M., Anvary M. (2009) CLONING OF PHENAZINE CARBOXYLIC ACID GENES OF ''FUSARIU MONILIFORME'' ANTAGONISTS BACTERIA IN ''ESHERICHIA COLI'' DH5α
 +
[http://www.agriculturaits.czu.cz/pdf_files/vol_42_4_pdf/kazempour.pdf ''AGRICULTURA TROPICA ET SUBTROPICA VOL. 42 (4) 157-161''

Revision as of 23:46, 4 October 2013



Phenazine


Overview

Figure 1: Molecular structure of phenazine.

Phenazines are water-soluble secondary metabolites secreted by some bacterial species. Almost all their properties can be attributed to their ability to undergo redox transformations. Some soil-born bacteria commonly produce phenazine-1-carboxylic acid ([http://www.ebi.ac.uk/chebi/chebiOntology.do?chebiId=CHEBI:62412 PCA])) against fungal root decease. (Niknejad Kazempour, [http://www.agriculturaits.czu.cz/pdf_files/vol_42_4_pdf/kazempour.pdf 2009]) Bacteria are also able to use this substance as a mediator for electron shuttling, which leads to an efficiency gain if a phenazine-producing strain is present in a MFC.
Problems arise with the different phenazine producing host strains. Most of them are classified as pathogens and therefore above the risk group, which is suitable for our lab. Moreover the team’s own risk assessment settles that no strain above the risk group one should be used. Hence, all experiments carried out in this section were soluble done with Pseudomonas fluorescence which is classified as risk group 1.
Initially PCA overproduction seemed to be a promising approach to achieve higher electron transfers. Because of the extremely complicated cloning procedure as well as the good advice of the consulted MFC expert Dr. Falk Harnisch, we abandoned this sub-project on early stages and searched for alternatives. We concentrated on an overexpression of riboflavin, which serves as an electrone shuttle itself and of the glycerol dehydrohenase gldA., which increases NADH concentration. (See sections riboflavin and gldA on our Wiki for more information). Still, quite a lot of work was invested into investigation of phenazine functions and synthesis in bacteria, of so we present a short theoretical overview of this topic. The only entry in the labjournal on phenazine is found here in Week 13.


Theory


Phenazines comprise a large group of heterocyclic nitrogen-containing substances, having a tricyclic ring in its core. They are substituted at different points around their rings, which alters water solubility and other properties. After discovery of naturally occurring phenazines, more than 6000 were chemically synthesized. (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/16719720 2006]). [http://en.wikipedia.org/wiki/Neutral_red Neutral red], a well-known pH-indicator, also belongs to this class.

Phenazine derivatives were one of the first secondary bacterial metabolites that were consequently studied since their discovery in 1859. The first substance of this family, a blue pigment of Pseudomonas aeroguinosa, was called [http://en.wikipedia.org/wiki/Pyocyanine «pyocyanine»] (short PYO). It had antimicrobial properties that raised further interest in these substances. Phenazines are produced by many bacterial species, but they are mostly studied in representatives of fluorescent pseudomonads. A single bacterial strain usually produces two or more species-specific phenazines, except Pseudomonas fluorescens 2-79, that is known to produce only PCA, a broad-spectrum antibiotic, which is a casual agent active against a variety of root pathogens (Mavrodi et al., [http://www.ncbi.nlm.nih.gov/pubmed/11591691 2001]).

Biologically phenazines are known for their broad-spectrum of antibiotical, antiparasaticand even anti-malaria properties (Handelsmann et al., [http://www.plantcell.org/content/8/10/1855.long 1996]). Consequently it was shown, that almost all properties of phenazines could be attributed to their ability to undergo redox transformations (Muller, [http://www.ncbi.nlm.nih.gov/pubmed/8541351 1995]). This discovery brought up the idea, that these molecules can be used as an electron shuttle in a MFC. Indeed, it was shown that external addition of phenazines, both PCA and PCN ([http://www.ebi.ac.uk/chebi/searchId.do?chebiId=62240 phenazine-1-carboxamide]) alike, causes a significant raise of current flow (The Hai Pham, [http://www.ncbi.nlm.nih.gov/pubmed/18688612 2008]). Surprisingly, a mixed culture of phenazine-secreting and phenazine non-secreting strains showed similar results, proving that phenazine can be used for redox cycling not only by its producer (Hernandez et al. [http://www.ncbi.nlm.nih.gov/pubmed/14766572 2004]).

Many strains of Pseudomonas fluorescens, a common soil inhabitant, are known to produce the phenazine compound PCA. The phenazine synthesis pathway was studied in detail. The seven-gene cluster [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 phzABCDEFG], 8505 bp long, was firstly identified in Pseudomonas fluorescens 2-79.

<p align="justify">Research on phenazine diversity and genetic evolution, (Mavrodi, [http://www.ncbi.nlm.nih.gov/pubmed/20008172 2010]) stated, that many species of fluorescent pseudomonas have the corresponding sequences and are able to produce phenazines, so we expected that similar genes are found in the Pseudomonas fluorescens 50090 that we obtained from [http://www.dsmz.de DSMZ].

Figure 2: Comparison of sequences between enzymes in phenazine cluster and other proteins with known function, according to research of [http://www.ncbi.nlm.nih.gov/pubmed/11562236 MacDonald et al. 2001.]

</p>


Genetic Approach

Figure 3: Phenazine synthesis cluster, comprising an autoinducer synthase (phzI) gene, positive regulator protein (phzR) and coding [http://www.ncbi.nlm.nih.gov/nuccore/L48616.1 phzABCDEFG] genes.


The coding cluster revealed seven forbidden restriction sites, making a cloning in accordance with the iGEM-rules an ultimate challengeUnfortunately, this step did never success. We varied different parameters and tried different enzymes, but we always saw a false priming and multiple amplificates. So we decided to look for applicable alternatives. Riboflavin, a natural mediator seemed an alternative of choice and our decision was rewarded by successful results.


Results

There are no practical results in this section. The project was left on the side after unsuccessful attempts to amplify the phenazine-coding fragment from Pseudomonas fluorescens sp. The only entry in the labjournal on phenazine is found here in Week 13.

References

  • Mavrodi D.V, Tomashow L.S. (2006) Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. [http://www.ncbi.nlm.nih.gov/pubmed/16719720 Annual review of Phytopathology 44:417– 445]
  • Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS (2001) Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. [http://www.ncbi.nlm.nih.gov/pubmed/11591691 J Bacteriol 183:6454–6465]
  • Handelsman, J. and E. V. Stabb (1996) Biocontrol of soilborne plant pathogens. [http://www.plantcell.org/content/8/10/1855.long Plant Cell 8(10): 1855-69.]
  • Vining L.C. (1990). Functions of secondary metabolites. [http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.44.100190.002143 Annu Rev Microbiol: 44:395-427]
  • Muller M. Scavenging of neutrophil-derived superoxide anion by 1-hydroxyphenazine, a phenazine derivative associated with chronic Pseudomonas aeruginosa infection: relevance to cystic fibrosis. (1995) [http://www.ncbi.nlm.nih.gov/pubmed/8541351 Biochim Biophys Acta: 1272(3):185-9.]
  • Pham T.H. (1998) Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation. [http://www.ncbi.nlm.nih.gov/pubmed/18688612 Appl Microbiol Biotechnology: 80(6):985-93.]
  • Hernandes M.E (2004) Phenazines and other redox-active antibiotics promote microbial mineral reduction [http://www.ncbi.nlm.nih.gov/pubmed/14766572 Appl Environ Microbiol.:70(2):921-8.]
  • McDonald M, Mavrodi DV, Thomashow LS, Floss HG. (2001). Phenazine biosynthesis in Pseudomonas fluorescens: branchpoint from the primary shikimate biosynthetic pathway and role of phenazine-1,6-dicarboxylic acid. [http://www.ncbi.nlm.nih.gov/pubmed/11562236 J. Am. Chem. Soc. 123:9459–60]
  • Mavrodi D.V. (2010) Diversity and evolution of the phenazine biosynthesis pathway. [http://www.ncbi.nlm.nih.gov/pubmed/20008172 Appl Environ Microbiol.:866-79.]
  • Niknejad Kazempour M., Anvary M. (2009) CLONING OF PHENAZINE CARBOXYLIC ACID GENES OF FUSARIU MONILIFORME ANTAGONISTS BACTERIA IN ESHERICHIA COLI DH5α

[http://www.agriculturaits.czu.cz/pdf_files/vol_42_4_pdf/kazempour.pdf AGRICULTURA TROPICA ET SUBTROPICA VOL. 42 (4) 157-161









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