Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose

Abstract

BackgroundNatural 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.ResultsThe 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.ConclusionsThis 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.

Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior

“…Repeated washing of the cell pellet led only to a marginal melanin extraction and no visible color shift of the cells, indicating that a large part of the formed melanin remained associated with the cells. This is in contrast to previously reported intracellular tyrosinase production, where the produced melanin has been found in the medium, and not associated to the cell surface1524, which has been explained through the excretion of a melanin precursor15.…”

Copper and Melanin Play a Role in Myxococcus xanthus Predation on Sinorhizobium meliloti

“…As shown in Figures 4A,B, the FT-IR spectra obtained of the solid brown pigment extracted in both samples were very similar. Moreover, these spectra exhibited several features that are identical to those described for bacterial melanins of E. coli (Chávez-Béjar et al, 2013;Mejia-Caballero et al, 2016), Lysobacter oligotrophicus (Kimura et al, 2014), Azotobacter chroococcum (Banerjee et al, 2014), Pseudomonas (Tarangini and Mishra, 2013), Streptomyces cyaneofuscatus (Al Khatib et al, 2018), Bacillus weihenstephanensis (Drewnowska et al, 2015), and soil bacteria (Tarangini and Mishra, 2014). Nevertheless, to further confirm that the pigment was melanin, the absorbance in UV-V of pigments extracted in NaOH from a pure culture of S. meliloti GR4 and a co-culture with M. xanthus following a modified version of the protocol described by Whittaker (1963) (see section "Materials and Methods" for details) was also determined.…”

Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation

“…The DAHP synthase has been regarded as a major ratelimiting step for synthesis of chorismate and its downstream aromatic compounds, and it has been a common strategy to increase the activity of this enzyme for improving the production of aromatic compounds (Báez-Viveros et al 2007;Chávez-Béjar et al 2013;Kikuchi et al 1997). In order to increase the supply of 4-hydroxybenzoate, a mutant of the aroG gene (encoding DAHP synthase), designated aroG fbr (G to A at 436 relative to the ATG start), was used to eliminate the feedback inhibition of DAHP synthase (Gosset et al 1996;Kikuchi et al 1997).…”