Characterization of melanin-overproducing transposon mutants of<i>Pseudomonas putida</i>F6

Nikodinovic-Runic, Jasmina; Martin, Leona B.; Babu, Ramesh; Blau, Werner J.; O’Connor, Kevin E.

doi:10.1111/j.1574-6968.2009.01716.x

Cited by 21 publications

(18 citation statements)

References 52 publications

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“…There is only one report on the production in a liquid medium of a melanin derived from homogentisic acid (pyomelanin). By employing the mutant Pseudomonas putida strain F6-HDO, 0.35 g/L of pyomelanin was produced [ 30 ]. In the case of allomelanins, this report constitutes the first example towards employing a microbial strain developed by metabolic engineering for the gram-level production of catechol melanin from glycerol.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli

et al. 2016

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BackgroundMelanins comprise a chemically-diverse group of polymeric pigments whose function is related to protection against physical and chemical stress factors. These polymers have current and potential applications in the chemical, medical, electronics and materials industries. The biotechnological production of melanins offers the possibility of obtaining these pigments in pure form and relatively low cost. In this study, Escherichia coli strains were engineered to evaluate the production of melanin from supplemented catechol or from glycerol-derived catechol produced by an Escherichia coli strain generated by metabolic engineering.ResultsIt was determined that an improved mutant version of the tyrosinase from Rhizobium etli (MutmelA), could employ catechol as a substrate to generate melanin. Strain E. coli W3110 expressing MutmelA was grown in bioreactor batch cultures with catechol supplemented in the medium. Under these conditions, 0.29 g/L of catechol melanin were produced. A strain with the capacity to synthesize catechol melanin from a simple carbon source was generated by integrating the gene MutmelA into the chromosome of E. coli W3110 trpD9923, that has been modified to produce catechol by the expression of genes encoding a feedback inhibition resistant version of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, transketolase and anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. In batch cultures with this strain employing complex medium with 40 g/L glycerol as a carbon source, 1.21 g/L of catechol melanin were produced. The melanin was analysed by employing Fourier transform infrared spectroscopy, revealing the expected characteristics for a catechol-derived polymer.ConclusionsThis constitutes the first report of an engineered E. coli strain and a fermentation process for producing a catechol melanin from a simple carbon source (glycerol) at gram level, opening the possibility of generating a large quantity of this polymer for its detailed characterization and the development of novel applications.

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli

et al. 2016

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“…Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time 1 , or oxidative stress disc diffusion assays 15 . These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates.…”

Section: Introductionmentioning

confidence: 99%

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Ketelboeter

Bardy

2015

JoVE

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Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

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“…Therefore, this mutation is expected to reduce HGA consumption by HGO. This result indicates that HGA is the allomelanin precursor in this mutant strain (Figure 3) (Nikodinovic-Runic et al, 2009). The synthesis of HGA originates from the L-tyrosine biosynthetic pathway.…”

Section: Random Mutagenesis For the Selection Of A Melanogenic Strainmentioning

confidence: 67%

Production of Melanins With Recombinant Microorganisms

Martínez

Martı́nez

Gosset

2019

Front. Bioeng. Biotechnol.

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The melanins constitute a diverse group of natural products found in most organisms, having functions related to protection against chemical and physical stresses. These products originate from the enzyme-catalyzed oxidation of phenolic and indolic substrates that polymerize to yield melanins, which include eumelanin, pheomelanin, pyomelanin, and the allomelanins. The enzymes involved in melanin formation belong mainly to the tyrosinase and laccase protein families. The melanins are polymeric materials having applications in the pharmaceutical, cosmetic, optical, and electronic industries. The biotechnological production of these polymers is an attractive alternative to obtaining them by extraction from plant or animal material, where they are present at low concentrations. Several species of microorganisms have been identified as having a natural melanogenic capacity. The development and optimization of culture conditions with these organisms has resulted in processes for generating melanins. These processes are based on the conversion of melanin precursors present in the culture medium to the corresponding polymers. With the application of genetic engineering techniques, it has become possible to overexpress genes encoding enzymes involved in melanin formation, mostly tyrosinases, leading to an improvement in the productivity of melanogenic organisms, as well as allowing the generation of novel recombinant microbial strains that can produce diverse types of melanins. Furthermore, the metabolic engineering of microbial hosts by modifying pathways related to the supply of melanogenic precursors has resulted in strains with the capacity of performing the total synthesis of melanins from simple carbon sources in the scale of grams. In this review, the latest advances toward the generation of recombinant melanin production strains and production processes are summarized and discussed.

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Characterization of melanin-overproducing transposon mutants ofPseudomonas putidaF6

Cited by 21 publications

References 52 publications

Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli

Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Production of Melanins With Recombinant Microorganisms

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