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Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose.

Chávez-Béjar MI, Balderas-Hernandez VE, Gutiérrez-Alejandre A, Martinez A, Bolívar F, Gosset G - Microb. Cell Fact. (2013)

Bottom Line: The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase.Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard.This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo, Postal 510-3, Cuernavaca, Morelos CP 62271, México. gosset@ibt.unam.mx.

ABSTRACT

Background: Natural aromatic polymers, mainly melanins, have potential and current applications in the cosmetic, pharmaceutical and chemical industries. The biotechnological production of this class of compounds is based on tyrosinase-dependent conversion of L-tyrosine and other aromatic substrates into melanins. The purpose of this work was to apply metabolic engineering for generating Escherichia coli strains with the capacity to synthesize an aromatic polymer from a simple carbon source.

Results: The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase. To direct the carbon flow from central metabolism into the common aromatic and the L-tyrosine biosynthetic pathways, feedback inhibition resistant versions of key enzymes were expressed in strains lacking the sugar phosphotransferase system and TyrR repressor. The expressed tyrosinase consumed intracellular L-tyrosine, thus causing growth impairment in the engineered strains. To avoid this issue, a two phase production process was devised, where tyrosinase activity was controlled by the delayed addition of the cofactor Cu. Following this procedure, 3.22 g/L of melanin were produced in 120 h with glucose as carbon source. Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard.

Conclusions: This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.

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Related in: MedlinePlus

Culture profiles carried out in shaken flasks with strains modified for L-tyrosine production from glucose (ca. 10 g/L) in medium lacking CuSO4 (tyrosinase is inactive without CuSO4). Symbols for strains: *W3110M, ■ W3110MG, ▲ W3110MGT, ▼VH33MGT, ♦ VH33MGTR and ● VH33MGTRK. Graphs represent the mean of two independent experiments.
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Figure 4: Culture profiles carried out in shaken flasks with strains modified for L-tyrosine production from glucose (ca. 10 g/L) in medium lacking CuSO4 (tyrosinase is inactive without CuSO4). Symbols for strains: *W3110M, ■ W3110MG, ▲ W3110MGT, ▼VH33MGT, ♦ VH33MGTR and ● VH33MGTRK. Graphs represent the mean of two independent experiments.

Mentions: Cultures for the production of melanin from L-tyrosine were performed at 30°C and 300 rpm agitation in 250 mL baffled shake flasks with 50 mL of M9 minimal salts medium [23] supplemented with 2 or 10 g/L of glucose, 0.1 mM IPTG, the required antibiotic for each strain and 0.4 g/L of L-tyrosine. 20 μg/mL CuSO4 were added at the beginning or at 16 h of culture time, as indicated in Figures 2 and 3. Kinetics for the production of melanin from glucose were carried out under the previously described conditions, but L-tyrosine was not added to culture media (Figure 3). Culture conditions for the production of L-tyrosine were the same as those employed for melanin production from glucose, except that CuSO4 was not added to the shake flasks culture medium (Figure 4). Bioreactor cultures for melanin production were started at an optical density at 600 nm of 1.0 from an inoculum grown overnight in M9 medium supplemented with 1% tryptone, 2 g/L of glucose and the required antibiotics for each strain. Cultures were performed using a working volume of 500 ml in 1-L stirred tank bioreactors model ADI 1010 (Applikon, The Netherlands). Culture medium employed was M9 supplemented with three pulses of glucose each one of 20 g/L. Air flow rate was maintained at 1 vvm and O2 partial pressure above 40%. Dissolved oxygen was measured with a polarographic oxygen probe (AppliSens; Applikon, Inc., Foster City, CA, USA). Based on previous studies, initial culture temperature was maintained at 32°C and then reduced to 30°C at the point when culture was in mid-exponential phase, corresponding to a biomass concentration between 5 and 6 g/L [9]. During these cultures, the pH was maintained at 7.0 and increased to 7.5 at mid-exponential phase. At this point, CuSO4 was added to a final concentration of 20 μg/mL.


Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose.

Chávez-Béjar MI, Balderas-Hernandez VE, Gutiérrez-Alejandre A, Martinez A, Bolívar F, Gosset G - Microb. Cell Fact. (2013)

Culture profiles carried out in shaken flasks with strains modified for L-tyrosine production from glucose (ca. 10 g/L) in medium lacking CuSO4 (tyrosinase is inactive without CuSO4). Symbols for strains: *W3110M, ■ W3110MG, ▲ W3110MGT, ▼VH33MGT, ♦ VH33MGTR and ● VH33MGTRK. Graphs represent the mean of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842659&req=5

Figure 4: Culture profiles carried out in shaken flasks with strains modified for L-tyrosine production from glucose (ca. 10 g/L) in medium lacking CuSO4 (tyrosinase is inactive without CuSO4). Symbols for strains: *W3110M, ■ W3110MG, ▲ W3110MGT, ▼VH33MGT, ♦ VH33MGTR and ● VH33MGTRK. Graphs represent the mean of two independent experiments.
Mentions: Cultures for the production of melanin from L-tyrosine were performed at 30°C and 300 rpm agitation in 250 mL baffled shake flasks with 50 mL of M9 minimal salts medium [23] supplemented with 2 or 10 g/L of glucose, 0.1 mM IPTG, the required antibiotic for each strain and 0.4 g/L of L-tyrosine. 20 μg/mL CuSO4 were added at the beginning or at 16 h of culture time, as indicated in Figures 2 and 3. Kinetics for the production of melanin from glucose were carried out under the previously described conditions, but L-tyrosine was not added to culture media (Figure 3). Culture conditions for the production of L-tyrosine were the same as those employed for melanin production from glucose, except that CuSO4 was not added to the shake flasks culture medium (Figure 4). Bioreactor cultures for melanin production were started at an optical density at 600 nm of 1.0 from an inoculum grown overnight in M9 medium supplemented with 1% tryptone, 2 g/L of glucose and the required antibiotics for each strain. Cultures were performed using a working volume of 500 ml in 1-L stirred tank bioreactors model ADI 1010 (Applikon, The Netherlands). Culture medium employed was M9 supplemented with three pulses of glucose each one of 20 g/L. Air flow rate was maintained at 1 vvm and O2 partial pressure above 40%. Dissolved oxygen was measured with a polarographic oxygen probe (AppliSens; Applikon, Inc., Foster City, CA, USA). Based on previous studies, initial culture temperature was maintained at 32°C and then reduced to 30°C at the point when culture was in mid-exponential phase, corresponding to a biomass concentration between 5 and 6 g/L [9]. During these cultures, the pH was maintained at 7.0 and increased to 7.5 at mid-exponential phase. At this point, CuSO4 was added to a final concentration of 20 μg/mL.

Bottom Line: The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase.Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard.This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo, Postal 510-3, Cuernavaca, Morelos CP 62271, México. gosset@ibt.unam.mx.

ABSTRACT

Background: Natural aromatic polymers, mainly melanins, have potential and current applications in the cosmetic, pharmaceutical and chemical industries. The biotechnological production of this class of compounds is based on tyrosinase-dependent conversion of L-tyrosine and other aromatic substrates into melanins. The purpose of this work was to apply metabolic engineering for generating Escherichia coli strains with the capacity to synthesize an aromatic polymer from a simple carbon source.

Results: The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase. To direct the carbon flow from central metabolism into the common aromatic and the L-tyrosine biosynthetic pathways, feedback inhibition resistant versions of key enzymes were expressed in strains lacking the sugar phosphotransferase system and TyrR repressor. The expressed tyrosinase consumed intracellular L-tyrosine, thus causing growth impairment in the engineered strains. To avoid this issue, a two phase production process was devised, where tyrosinase activity was controlled by the delayed addition of the cofactor Cu. Following this procedure, 3.22 g/L of melanin were produced in 120 h with glucose as carbon source. Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard.

Conclusions: This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.

Show MeSH
Related in: MedlinePlus