A Yarrowia lipolytica Strain Engineered for Pyomelanin Production.

Tristan Rossignol,Jean-Marc Nicaud,Olivier Rué,Djamila Onésime,Macarena Larroude

doi:10.3390/microorganisms9040838

Abstract

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Highlights

Melanins are a heterogeneous group of polymeric pigments that are widely found in nature, in organisms ranging from microbes to humans
We looked for the following compounds of interest: Ehrlich metabolites—2-(4-hydroxyphenyl)ethanol (4OH2PE), 4-hydroxyphenylacetic acid (4OHPAA), phenylethanol (2PE), and phenylacetic acid (PAA); aromatic amino acid (AAA); and homogentisic acid (HGA)
Previous research has shown that Y. lipolytica can produce a brown pigment, found to be a polymer composed of a core of tyrosine-derived aromatic residues [13]

Summary

Introduction

Melanins are a heterogeneous group of polymeric pigments that are widely found in nature, in organisms ranging from microbes to humans. Pyomelanin in particular is a brown pigment that is produced via the oxidation of homogentisic acid (HGA) synthesized from tyrosine [1]. Apte et al [9,18] enhanced melanin as an electron exchanger, the resulting melanin was used to synthesize silver and gold production in Y. lipolytica by culturing the yeast in the presence of L-DOPA. Nanoparticles are useful paint additives antifungal its ability to Silver act as an electron exchanger, the resulting melanin because was usedoftotheir synthesize properties. Despite this body of work, is how Y. lipolytica silver and gold nanoparticles.

Schematic representation of the pyomelanin

Strain and Plasmid Construction

Strain Sequencing and Integration Site Identification

Media and Culture Conditions

HPLC Analysis

Pyomelanin Purification

Production of a Brown Pigment by Yarrowia Lipolytica

Characterization of the Brown Pigment

Characterization and Improvement the Pyomelanin