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. 2021 Apr 14;9(4):838.
doi: 10.3390/microorganisms9040838.

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production

Macarena Larroude  1 , Djamila Onésime  1 , Olivier Rué  2   3 , Jean-Marc Nicaud  1 , Tristan Rossignol  1
Affiliations

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production

Macarena Larroude et al. Microorganisms. .

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.

Keywords: Yarrowia lipolytica; aromatic amino acids; chassis strain; pyomelanin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the pyomelanin biosynthesis pathway. PHE: phenylalanine; TYR: tyrosine; Tyr-AT: tyrosine aminotransferase; 4-HPPA: 4-hydroxyphenylpyruvic acid, 4HPPD: 4-hydroxyphenylpyruvic acid dioxygenase; HGA: homogentisic acid.
Figure 2
Figure 2
Production of pyomelanin precursors. Top: Concentrations of tyrosine (TYR), phenylalanine (PHE), homogentisic acid (HGA), phenylethanol (2PE), phenylacetic acid (PAA), 2-(4-hydroxyphenyl)ethanol (4OH2PE), and 4-hydroxyphenylacetic acid (4OHPAA) in the supernatants of the three strains at 5 days of culture. Values correspond to average of 2 replicates. Middle: Strain genotypes. Bottom: Images illustrating pigmentation levels in the cultures at 25 days of growth.
Figure 3
Figure 3
(A) Changes in pigment levels over time and in response to tyrosine (TYR) or phenylalanine (PHE) supplementation. (B) Inhibitory effect of ascorbic acid on pigment production. (C) Inhibitory effect of low pH on pigment production, due to the absence of buffer in the medium. All cultures were performed using the strain JMY8032.
Figure 4
Figure 4
Effect of 4HPPD disruption and overexpression on brown pigment production in the strain JMY8032 in YPD medium after 4 days of culture.
Figure 5
Figure 5
Production of homogentisic acid and Ehrlich metabolites (in g/L) by the four study strains (JMY8032, JMY8131, JMY8178, and JMY8208) in YNB medium. The individual quantities of 2-4OHPE, 4OHPAA, 2PE, and PAA were summed to obtain the total quantity of Ehrlich metabolites. 7D: 7 days of culture; 14D: 14 days of culture.
See this image and copyright information in PMC

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